Optimization of enzyme replacement therapy for treatment of homocystinuria

ABSTRACT

The present invention provides a method of PEGylating a human truncated cystathionine β-synthase protein containing a mutation of a cysteine to a serine at amino acid position 15 (htCBS C15S). The htCBS C15S was PEGylated with one of 5 kDa, 10 kDa, or 20 kDa NHS ester PEG molecules. In-process monitoring of the PEGylation process was used in the method to reduce levels of unPEGylated htCBS C15S and htCBS C15S with insufficient PEGylation. Administration of the PEGylated htCBS C15S had efficacy throughout the course of treatment for homocystinuria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/605,918 filed Oct. 17, 2019, now U.S. Pat. No. 11,324,811, which is anational stage application of International Patent Application No.PCT/US2018/027854 filed Apr. 17, 2018, which claims priority to U.S.provisional patent application 62/486,246 filed Apr. 17, 2017, each ofwhich is herby incorporated by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file, entitled790116_432C1_SEQUENCE_LISTING.txt, was created on Apr. 4, 2022, and is10,921 bytes in size. The information in electronic format of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to characterizing PEGylated human truncatedcystathionine beta-synthase (htCBS) conjugates to identify theirsuitability for reproducible manufacturing and further clinicaldevelopment as an enzyme replacement therapy for classicalhomocystinuria.

BACKGROUND OF THE INVENTION

Human cystathionine beta-synthase (CBS, KEGG enzyme identifier: EC4.2.1.22) is an important enzyme of the sulfur amino acid metabolism,whose activity determines either recycling of the sulfur in themethionine (Met) cycle or its diversion into synthesis of cysteinethrough the transsulfuration pathway. See, Finkelstein, J. D., J. Nutr.Biochem. 1990, 1, 228-237, which is hereby incorporated by reference inits entirety. The critical intermediate of sulfur amino acid metabolismis the non-proteinogenic amino acid homocysteine (Hcy), which can beeither converted back to Met by an action of Met synthase or betainehomocysteine methyltransferase or redirected by CBS to cysteinebiosynthesis via the transsulfuration pathway. CBS is a pyridoxal5′-phosphate-dependent homotetrameric hemeprotein, which catalyzes thecondensation of Hcy with serine forming cystathionine (Cth) and water.Cth is subsequently hydrolyzed by the action of cystathioninegamma-lyase to cysteine (Cys), alpha-ketobutyrate and ammonia, whatcompletes the conversion of the essential amino acid Met to Cys.Activity of CBS, and thus the flux of sulfur through thetranssulfuration pathway, is attenuated by the autoinhibitory functionof the enzyme's C-terminal regulatory domain. Binding ofS-adenosylmethionine to this domain results in a conformationalrearrangement of the regulatory domain, subsequent release of theautoinhibitory block and 4-5-fold activation of the enzyme. See,Ereno-Orbea et al., Proc Natl Acad Sci USA 2014, 111, (37), E3845-52,which is hereby incorporated by reference in its entirety. Similar levelof CBS activation can be achieved by removal of the C-terminalregulatory domain and consequent formation of a dimeric, permanentlyactivated human truncated enzyme (htCBS). See, Kery et al., Arch BiochemBiophys 1998, 355, (2), 222-32, which is hereby incorporated byreference in its entirety.

Lack of CBS activity results in classical homocystinuria (HCU), anautosomal recessively inherited rare metabolic disorder. See, Mudd etal., Disorders of transsulfuration. In The Metabolic and Molecular Basesof Inherited Disease, 8 ed.; Scriver, C. R.; Beaudet, A. L.; Sly, W. S.;Valle, D.; Childs, B.; Kinzler, K.; Vogelstein, B., Eds. McGraw-Hill:New York, 2001; pp 2007-2056 which is hereby incorporated by referencein its entirety. The inactivation of CBS, chiefly due to the presence ofmissense pathogenic mutations, yields severe elevation of Hcy, Met, andS-adenosylhomocysteine and conversely lack of Cth and decrease of Cys inplasma samples of HCU patients. These biochemical sequelae areaccompanied with connective tissue disorders, such as dislocated opticlenses, long and thin limbs and osteoporosis, thromboembolism, cognitiveimpairment and other clinical symptoms. See, Mudd et al. Presenttreatment of HCU is mostly symptomatic aimed at reduction of Hcyconcentration in the plasma and tissues by protein-restricted diet withMet-free L-amino acid supplementation. See, Komrower et al., Arch DisChild 1966, 41, (220), 666-71, which is hereby incorporated by referencein its entirety. This dietary intervention is often combined withbetaine, which promotes the remethylation of Hcy back to Met. See,Smolin et al. J Pediatr 99(3), 467-472 (1981), which is herebyincorporated by reference in its entirety. In addition, supplementationwith the pyridoxal 5′-phosphate precursor pyridoxine (vitamin B6) wasshown to be effective in roughly 50% of HCU patients by stimulating theresidual CBS activity and thus relieving the clinical manifestation andrelaxing the dietary regime. See, Barber et al. J Pediatr 75(3), 463-778(1969), which is hereby incorporated by reference in its entirety.Although Met restriction is generally effective in lowering plasma Hcylevels, a compliance of the HCU patients with the diet is quite poorwith consequent manifestation of the symptoms. See, Walter et al., Eur.J. Pediatr. 1998, 157 (Suppl 2), S71-S76, which is hereby incorporatedby reference in its entirety. Thus, there is a need in the art for analternative therapy that would not require a stringent diet but wouldachieve similar reduction of Hcy levels and prevent onset and/orescalation of the clinical symptoms.

Enzyme replacement therapy (ERT) for HCU has recently been describedusing PEGylated htCBS. See, Bublil et al., J Clin Invest 2016, 126, (6),2372-84 and International application PCT/2016/061050, which are herebyincorporated by reference in their entireties. PEGylation is conjugationof protein of interest to one or more polyethylene glycol (PEG) moietiesor molecules and is a well-recognized and accepted strategy to alterpharmacokinetic profiles of a variety of drugs to improve theirtherapeutic potential. See, Fishburn et al., J Pharm Sci 2008, 97, (10),4167-83, which is hereby incorporated by reference in its entirety.Unmodified htCBS exhibited rapid clearance from the circulation of themouse model of the disease (elimination half-life after IVadministration was 2.7 h). PEGylation of htCBS has been observed tolengthen the elimination half-life of PEGylated htCBS after IVadministration by 6-11-fold, improve the plasma metabolic profile ofmurine homocystinuria in “Human Only” (HO) mice, and rescue the CBSknock-out (KO) mice from neonatal lethality by preventing liver damage.See, Bublil et al., J Clin Invest 2016, 126, (6), 2372-84; each of whichis herein incorporated by reference in its entirety.

There is a long-felt need in the art to design and characterize aPEGylated htCBS conjugate that exhibits high reproducibility and controlduring manufacturing as well as having long-term efficacy. In previousstudies, introduction of the C15S point mutation to htCBS was observedto diminish formation of protein aggregates and higher order oligomersand improved PEGylation profile for 20 kDa linear maleimide-activatedPEG molecule. See, Bublil et al. Various htCBS C15S conjugate PEGylatedwith either maleimide (MA) or N-hydroxysuccinimide (NHS) ester-activatedPEG molecule (further referred as PEG-htCBS C15S) are characterizedherein for their potency to correct metabolic profile of murinehomocystinuria after a single administration or multiple rounds ofrepeated administration interrupted with washout periods. The PEG-htCBSC15S conjugates were ranked based on their pharmacodynamics. Inaddition, insight was gained into reproducibility and consistency ofPEG-htCBS preparation and characterization of the sites modified by thePEG molecules. Assays were identified to monitor and evaluate the extentof htCBS C15S PEGylation.

SUMMARY OF THE INVENTION

Various embodiments of the invention herein provide a method ofPEGylating a human truncated cystathionine β-synthase (CBS) proteincontaining a mutation of a cysteine to a serine at amino acid position15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S), the methodcomprising: (a) conjugating the htCBS C15S with one or a plurality ofNHS ester PEG molecules in solution at a molar excess of the NHS esterPEG molecules up to about 40-fold to create a batch, wherein theplurality of NHS ester PEG molecules are 5 kDa, 10 kDa, or 20 kDa NHSester PEG molecules; (b) comparing a retention time from achromatographic profile from Size Exclusion Chromatography-HighPerformance Liquid Chromatography (SEC-HPLC) analysis of the batch to aretention time from a chromatographic profile from SEC-HPLC analysis ofhtCBS C15S with acceptable PEGylation to identify insufficientPEGylation, wherein the batch with insufficient PEGylation has aretention time greater than the retention time of the htCBS C15S withacceptable PEGylation; and (c) adding additional NHS ester PEG moleculesto the batch to reduce the amount of insufficient PEGylation in thebatch, thereby PEGylating the htCBS C15S.

Various embodiments of the invention herein provide a method ofPEGylating a human truncated cystathionine β-synthase (CBS) proteincontaining a mutation of a cysteine to a serine at amino acid position15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S), the methodcomprising: (a) conjugating the htCBS C15S with one or a plurality ofNHS ester PEG molecules in solution at a molar excess of the NHS esterPEG molecules less than about 40-fold to create a batch, wherein theplurality of NHS ester PEG molecules are 5 kDa, 10 kDa, or 20 kDa NHSester PEG molecules; (b) comparing a retention time from non-reducedcapillary electrophoresis (NR-CE) analysis of the batch to a retentiontime from NR-CE analysis of htCBS C15S with acceptable PEGylation toidentify insufficient PEGylation, wherein the batch with insufficientPEGylation has a retention time less than the retention time of thehtCBS C15S with acceptable PEGylation; and (c) adding additional NHSester PEG molecules to the batch to reduce the amount of insufficientPEGylation in the batch, thereby PEGylating the htCBS C15S.

In certain embodiments, NHS ester PEG molecules are present in a molarexcess of about 10-fold. In certain embodiments, the NHS ester PEGmolecules are present in a molar excess of about 20-fold. Alternatively,the NHS ester PEG molecules are present in a molar excess less than20-fold, less than 10-fold, less than 5-fold, or less than 2-fold.Certain embodiments provide that the plurality of NHS ester PEGmolecules consists of about 5 kDa NHS ester (5NHS) PEG molecules. Incertain embodiments, the plurality of NHS ester PEG molecules consistsof about 20 kDa NHS ester (20NHS) PEG molecules. In certain embodiments,the plurality of NHS ester PEG molecules is less than about 20 kDa. Forexample, the plurality of NHS ester PEG molecules consists of about 10kDa NHS ester (10NHS) PEG molecules. In certain embodiments, htCBS C15Swith insufficient PEGylation comprises fewer than 15, fewer than 10,fewer than 5, or fewer than 1 PEGylated amino acids. In certainembodiments, htCBS C15S with acceptable PEGylation comprises at least 1PEGylated amino acid.

In certain embodiments, the retention time of the batch of htCBS C15Swith insufficient PEGylation is greater than about 9.50 minutes, about9.75 minutes, about 10.00 minutes, and about 10.25 minutes. In certainembodiments, the retention time of the batch of htCBS C15S withinsufficient PEGylation is between about 9.60 minutes and about 9.70minutes. In certain embodiments, the retention time htCBS C15S withacceptable PEGylation is within a range from about 9.50 to about 9.60minutes. In certain embodiments, the retention time htCBS C15S withacceptable PEGylation is less than about 9.53 minutes. For example, aPEGylated htCBS C15S with insufficient PEGylation comprises zero (0)PEGylated amino acids.

Various embodiments of the invention herein provides a method ofmaintaining efficacy of a treatment for homocystinuria in a subjectthroughout the duration of the treatment, the method including the stepsof: administering to the subject a therapeutically effective amount ofhuman truncated cystathionine β-synthase (CBS) protein containing amutation of a cysteine to a serine at amino acid position 15 of SEQ IDNO: 1 or a variant thereof (htCBS C15S) PEGylated using the methodabove. In certain embodiments, the efficacy of the treatment ismaintained for up to 2 weeks, up to 1 month, up to 6 months, or up to 1year. Alternatively, the efficacy of the treatment is maintained forlonger than 1 year.

Various embodiments of the invention herein provide a method of reducingthe level of homocysteine (Hcy) in a subject with homocystinuria, themethod including: administering to the subject a therapeuticallyeffective amount of human truncated cystathionine β-synthase (CBS)protein containing a mutation of a cysteine to a serine at amino acidposition 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S) PEGylatedusing the method above. In certain embodiments, the level of Hcy isreduced in a range selected from the group of ranges selected from about5% to about 10%, about 10% to about 20%, about 20% to about 300, about30% to about 40%, about 40% to about 50%, about 60% to about 70%, about70% to about 80%, about 80% to about 90%, and about 90% to about 100%.

Various embodiments of the invention herein provide a method ofincreasing the level of cysteine (Cys) in a subject with homocystinuria,the method including the steps of: administering to the subject atherapeutically effective amount of human truncated cystathionineβ-synthase (CBS) protein containing a mutation of a cysteine to a serineat amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBSC15S) PEGylated using the method above. In certain embodiments, thevariant shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity with SEQ ID NO: 1. In certainembodiments, a therapeutic effect resulting from the administering stepis maintained for at least 6 hours, at least 12 hours, at least 18hours, or at least 24 hours.

Various embodiments of the invention herein provide a pharmaceuticalcomposition including a human truncated cystathionine β-synthase (CBS)protein containing a mutation of a cysteine to a serine at amino acidposition 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S) PEGylatedusing the method above; and a pharmaceutically acceptable carrier,diluent, or excipient. In certain embodiments, the variant in thepharmaceutical composition shares at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows non-reduced capillary electrophoresis (NR-CE) profiles ofmultiple 20NHS PEG-htCBS C15S batches compared to an unmodified htCBSC15S protein.

FIG. 2 shows chromatographic separation of 400MA PEG-htCBS C15S specieson DEAE Sepharose using a shallow salt gradient (5%, 10-110 mM NaCl).The straight line represents absorbance at 280 nm, while the peaks showchanges in conductivity of the mobile phase. Both lines were generatedusing BIO-RAD® Biologic LP chromatographic system with BIO-RAD® BiologicLP Data View Software version 1.03. “S” represents the time point of thestart of the pump and recording by the software program, and “E”represents the time point of the end of operation of the pump andrecording by the software program. “M” represents a manually designatedpoints of the beginning of the monitoring of Pool 1 and Pool 2 asindicated by the labels in the figure.

FIG. 3A-3C show maleimide PEGylation mapping using a Lys-C digest. Thesefigures show reduced alkylated LC-MS peptide maps after a Lys-C digestof unmodified htCBS C15S as a reference (FIG. 3A), homoPEGylated 400MAPEG-htCBS C15S (P1) (FIG. 3B), and hemiPEGylated 400MA PEG-htCBS C15S(P2) (FIG. 3C) proteins.

FIG. 4A-4C show maleimide PEGylation mapping using a NTCB digest. Thesefigures show LC-MS peptide maps after NTCB treatment of unmodified htCBSC15S as a reference (FIG. 4A), homoPEGylated 400MA PEG-htCBS C15S (P1)(FIG. 4B), and hemiPEGylated 400MA PEG-htCBS C15S (P2) (FIG. 4C) proteinsamples.

FIG. 5 shows CBS specific activity of various NHS PEG-htCBS C15Sconjugates compared to unmodified htCBS C15S. Bars represent the averagefrom 3 separate measurements and error bars designate standard error ofthe means (SEMs).

FIG. 6 shows evaluation of 5NHS PEG-htCBS C15S in a washout experiment.Plasma levels of Hcy (squares), Cth (circles) and Cys (diamonds) afterrepeated administration of 5NHS PEG-htCBS C15S in HO mice (SC, 7.5mg/kg). Points represent an average value from individual HO mice (n=6)and error bars indicate SEMs.

FIG. 7A-7B show body weight (FIG. 7A) and weight gain (FIG. 7B) of 20NHSPEG-htCBS C15S-treated CBS KO male (solid line and circles) and female(dashed line and circles) mice compared to PBS-injected+/−males (solidline and squares) and females (dashed line and squares). The number ofsubjects in each group is indicated in the plots. Mice receivedsubcutaneous injections of 7.5 mg/kg 3 times per week from day 2 of age.

FIG. 8A-8B show effects of a long-term treatment of the I278T mice with20NHS PEG-htCBS C15S, which sustains improved plasma sulfur amino acidprofiles. FIG. 8A shows metabolite concentrations in weaned (day 21)I278T mice (n=3) that were treated 3× a week with 20NHS PEG-htCBS C15S(SC, 7.5 mg/kg) for about 9 months. Day 21 shows plasma levels of Hcy(solid line and squares), Cth (dashed line and circles), and Cys (dashedline and diamonds) prior the treatment commencement, while days 28 and63 illustrate the plasma levels 72 hours after the 3rd and 18thinjection, respectively (i.e. weekend washout). All subsequent timepoints show plasma sulfur amino acids 24 hours after dosing. FIG. 8Bshows results for the adult, fully symptomatic I278T mice (n=10)injected 3 times per week with 20NHS PEG-htCBS C15S (SC, 7.5 mg/kg) fromage of 26 weeks for a period of about 6 months. Day 182 shows initiallevels prior treatment and all the subsequent time points representplasma metabolites 24 hours after dosing. Symbols represent an averagevalue from individual mice and error bars indicate standard error of themeans (SEMs).

FIG. 9A-9C show pharmacokinetics of 20NHS PEG-htCBS C15S after a singledose administration and after a repeated administration to SpragueDawley rats. FIG. 9A shows a semi-log plot of CBS specific activity inplasma of male (black squares, n=11 each group) and female rats (graycircles, n=8 each group) after a single SC administration of 8 mg/kg(solid lines) or 24 mg/kg 20NHS PEG-htCBS C15S (dashed lines). CBSspecific activity in plasma of male (black, n=8) and female rats (gray,n=8) after repeated SC injections of 4 mg/kg (FIG. 9B) or 24 mg/kg 20NHSPEG-htCBS C15S (FIG. 9C). The gray arrows designate a dosing time pointfor a total of 9 doses administered every 2 days. Data points in allplots represent average values and error bars show SEMs.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Homocystinuria (HCU) due to loss of CBS causes accumulation ofhomocysteine (Hcy) and depletion of cysteine (Cys). There is a long feltneed in the art for an optimized targeted treatment, which would addressthe underlying enzyme deficiency, thus the development of an enzymereplacement therapy based on PEGylated human truncated CBS (PEG-htCBS)has been explored herein. Attenuation of potency was observed, whichwarranted screening of several different PEG-htCBS C15S conjugates. ThePEG-htCBS C15S conjugates were ranked based on their efficacy to correctplasma metabolite profile of murine homocystinuria after repeatedadministrations interrupted with washouts. CBS was observed to couplewith maleimide PEG molecules inconsistently. In contrast, the PEG-htCBSC15S conjugate with 20 kDa linear NHS-PEG showed very little loss ofpotency likely due to reproducible PEGylation resulting in speciesmodified with an average of 5 PEGylation sites per peptide. In addition,assays were developed to monitor the extent of CBS PEGylation. Theseresults identified the PEG-htCBS C15S conjugate of those analyzed mostsuitable for manufacturing and clinical development.

An integral part of any novel therapeutic development effort, whether itis a small molecule drug or a biologic, is the selection, optimizationand characterization of a lead candidate to enter the clinical trials.Although effective, treatment of HCU to reduce methionine (Met) intakethrough dietary restrictions is very difficult to follow and results inserious patient compliancy issues. See, Walter et al., Eur. J. Pediatr.1998, 157 (Suppl 2), S71-S76, which is hereby incorporated by referencein its entirety. The therapeutic goal of the currently availabletreatments for HCU, i.e., a restricted diet, pyridoxine, and/or betainesupplementation, is to reduce or normalize plasma Hcy levels becausefluctuations and high levels of Hcy are often associated with seriouscomplications, particularly thromboembolic events. See, Morris et al.,Guidelines for the diagnosis and management of cystathioninebeta-synthase deficiency. J Inherit Metab Dis 2016, which is herebyincorporated by reference in its entirety. Development of an alternativetreatment, such as ERT, would reduce or eliminate reliance on dietaryrestrictions, which would allow HCU patients and their families accessto a better quality of life. To achieve this therapeutic goal (i.e. thereduction or normalization of plasma levels of Hcy), the htCBS C15Svariant has been previously modified with 20 kDa linearmaleimide-activated PEG molecule and proof of concept was provided usingmurine models of HCU. See, Bublil et al., J Clin Invest 2016, 126, (6),2372-84 and International application PCT/2016/061050, which are herebyincorporated by reference in their entireties.

There is a trend in PEGylation of therapeutic macromolecules fromheterogeneous mixtures of conjugate isomers towards a single modifiedform, such as characterization/identification of the product andreproducibility of the manufacturing process. See, Pasut et al., JControl Release 2012, 161, (2), 461-72, which is hereby incorporated byreference in its entirety. Modification of accessible Cys residues usingmaleimide PEG molecules is one of the most commonly used approaches forsite selective PEGylation. Despite the mutagenesis of the mostaccessible cysteine residue 15 into serine (C15S) in htCBS, multiplePEGylated species were observed in addition to a fraction of anunmodified enzyme. See, Frank et al., Biochemistry 2006, 45, (36),11021-9, which is hereby incorporated by reference in its entirety.Although the individual species were chromatographically separable, suchoperation would certainly result in substantial losses and/or wouldlikely render the process economically non-viable. Maleimide PEGylationof htCBS C15S was mapped to a peptide containing residues C272 and C275with the former residue being a primary site for modification.Mutagenesis of either Cys residue would most likely increasedreproducibility of PEGylation; however, substantial reduction in hemesaturation and catalytic activity was anticipated. See, Taoka et al.,Biochemistry 2002, 41, (33), 10454-61, which is hereby incorporated byreference in its entirety.

Similar strategy for PEGylation was employed in the development of anERT for phenylketonuria based on phenylalanine ammonia-lyase (PAL).Unlike CBS described herein, the PAL is a tetrameric non-mammalianenzyme originating from cyanobacterial and fungal species. See,Sarkissian et al., Proc Natl Acad Sci USA 2008, 105, (52), 20894-9,which is hereby incorporated by reference in its entirety. Therefore,immunogenicity of such a foreign protein represented a substantialconcern and various PEGylation protocols were tested with PAL. See,Gamez et al., Molecular therapy: the journal of the American Society ofGene Therapy 2005, 11, (6), 986-9, which is hereby incorporated byreference in its entirety. PAL was conjugated with NHS ester PEGsranging from a linear 5 kDa up to a branched 40 kDa in size with linear20 kDa PEG-PAL conjugates demonstrating increased performance andmasking immunogenicity the best. Reduction of the PEG-PAL immunogenicitywas directly proportional to the number of PEGs attached to the PALsubunit with gel-based estimate of 6-8 PEGs per PAL monomer suppressingthe immune response most efficiently. See, Gamez et al.

When PEG-PAL was administered to the PKU mouse model, plasmaphenylalanine levels were brought down to zero in short-term study andafter overcoming an initial immune response also in a long-term study.See, Sarkissian et al., Proc Natl Acad Sci USA 2008, 105, (52), 20894-9,which is hereby incorporated by reference in its entirety. The Hcymetabolism and pathophysiology need to be taken into an account. Unlikephenylalanine, Hcy is a redox-active thiol group-containing moleculeexisting in plasma in a variety of chemical forms (as fractions of totalHcy pool): thiolactone (<1%), free reduced (about 2%), disulfide (about18%), and protein-bound (about 80%). Refsum et al., Clin Chem 1985, 31,624-628; Rasmussen et al. Ann Clin Biochem 2000, 37 (Pt 5), 627-48;Jakubowski, H., Anal Biochem 2002, 308, (1), 112-9, each of which arehereby incorporated by reference in their entireties.

Free Hcy is increased substantially to a 10-25% of total plasma Hcy inHCU. See, Mansoor et al., Metabolism 1993, 42, 1481-1485. In addition,Hcy, in homocystinuria. substantially replaces Cys bound to plasmaalbumin, what may contribute to decreased levels of plasma Cys found inHCU. See, Bar-Or et al., Biochem Biophys Res Commun 2004, 325, (4),1449-53, which is hereby incorporated by reference in its entirety.Albumin is synthesized exclusively in the liver and its plasmaconcentration of 33-52 g/l in healthy human represents only about 40%,while the remaining about 60% of albumin stays in extravascular space.See, Boldt et al., Br J Anaesth 2010, 104, (3), 276-84, which is herebyincorporated by reference in its entirety. In theory, only a free Hcyrepresents a substrate for CBS. Thus, the 50-80% decrease of total Hcyin HO mice upon continuous administration of 20NHS PEG-htCBS C15S may beexplained by shifting the balance of metabolites in favor of free Hcycompared to other forms (disulfides and protein-bound). See, Bublil etal., J Clin Invest 2016, 126, (6), 2372-84 and International applicationPCT/2016/061050, which are hereby incorporated by reference in theirentireties. 20NHS PEG-htCBS C15S in circulation serves as a “sink” forexcess of Hcy to improve the Hcy plasma levels. Possible reasons forfailing to normalize levels could be the constant flux of intracellularHcy (particularly from the liver as Hcy bound to a newly synthesizedalbumin), dynamic balance between intra- and extra-vascular Hcy pools(mediated mostly by albumin), and homocystinuria-induced pro-oxidativeenvironment favoring disulfides and protein-bound forms of total Hcypool. Nevertheless, recently published guidelines for diagnosis andmanagement of HCU recommend keeping the plasma total of Hcy below 100 μMto prevent progress and/or occurrence of serious complications. See,Morris et al., Guidelines for the diagnosis and management ofcystathionine beta-synthase deficiency. J Inherit Metab Dis 2016, whichis hereby incorporated by reference in its entirety. Values areextrapolated from the results herein from homocystinuric HO mice fedwith standard unrestricted chow containing 4-5 g Met per kg of diet(corresponds to 18-19% of protein). Administration of 20NHS PEG-htCBSC15S to human HCU patients results in guideline-recommended total Hcylevels in plasma without any additional treatment. Based on the resultsherein, a GLP-toxicology (GLP-tox) study in humans using 20NHS PEG-htCBSC15S as an ERT for HCU has been initiated.

II. Methods of Characterizing Pegylated htCBS C15S

A. Extent of PEGylation of htCBS C15S (PEG-htCBS C15S)

In certain embodiments, the extent of PEGylation of htCBS C15S may beused to define acceptance criteria for individual PEG-htCBS C15S. Insome embodiments, the PEG molecule is ME-200GS (also known as 20NHS).

In certain embodiments, size exclusion chromatography (SEC)-highperformance liquid chromatography (HPLC) is used to compare a batch ofPEG-htCBS C15S during processing to unmodified htCBS C15S.Insufficiently PEGylated htCBS batches are identified by longerretention times than the retention times for acceptable PEGylation. Incertain embodiments, a retention time of greater than about 9.5 minutes,about 9.75 minutes, about 10 minutes, and about 10.25 minutes indicatesinsufficient PEGylation (<1 PEG/protein or <5 PEGs/protein). Forexample, a retention time between 9.6 and 9.7 minutes, such as 9.67minutes, may indicate insufficient PEGylation.

In certain embodiments, a retention time of less than about 9.6 minutes,about 9.4 minutes, about 9.2 minutes, and about 9 minutes was determinedto indicate acceptable PEGylation (at least 1 PEG/protein). For example,a retention time between 9.5 and 9.6 minutes, such as 9.53 minutes, mayindicate acceptable PEGylation.

In some embodiments, additional PEG molecules may be added to thereaction mixture for insufficiently PEGylated batches to potentiallyrescue the extent of PEGylation. The comparison by SEC-HPLC may beperformed multiple times during the PEGylation process. Alternatively,the comparison by SEC-HPLC may be performed once during the PEGylationprocess.

In certain embodiments, non-capillary electrophoresis is used toquantify multiple PEG-htCBS C15S species. Insufficiently PEGylatedbatches of PEG-htCBS C15S were identified by the presence of unmodifiedenzyme and an increased occurrence of low molecular weight PEG-htCBSC15S species. In certain embodiments, liquid chromatography-massspectrometry (LC-MS) is used to estimate the number of PEG molecules perpeptide. In certain embodiments, at least seven PEGylated lysines areobserved to be modified in an PEGylated htCBS C15S with acceptablePEGylation. For example, K25, K30, K211, K247, K271, K405, and K406 ofhtCBS C15S may be PEGylated. The lysines may have varying degrees ofPEGylation. More specifically, K25 and K30 of htCBS C15S may bePEGylated to the full extent, while other lysines may be partiallyPEGylated.

In certain embodiments, acceptable PEGylation is defined as about 5PEGylated sites per htCBS C15S protein. Alternatively, acceptablePEGylation may be selected from at least one range of between 1-5PEGylated sites, between 3-7 PEGylated sites, and between 5-9 PEGylatedsites per htCBS CBS protein. For example, acceptable PEGylation of htCBSC15S may include one PEGylated lysine per protein. Alternatively,acceptable PEGylation of htCBS C15S may include about 5 PEGylatedlysines per protein.

B. Optimizing PEG Conjugation

Various approaches such as reduction of accessible cysteines withtris(2-carboxyethyl)phosphine (TCEP) prior to PEGylation to enhance theyield of the modified enzyme or multiple PEGylation schemes to gainconsistency in the process have been previously observed to partiallyimprove the consistency of PEGylation.

Maleimide PEGylation of htCBS may be site-specific. In certainembodiments, htCBS C15S is first PEGylated at C272 during maleimidePEGylation. Alternatively, htCBS C15S is PEGylated at C272 and C275during maleimide PEGylation.

Alternatively, htCBS C15S may be PEGylated by at least one of 5, 10, or20 kDa linear NHS ester PEG molecules. In certain embodiments,PEGylation of htCBS C15S by a NHS ester PEG molecule results in areproducible pattern. In certain embodiments, PEGylation is performedwith up to 20-fold excess of PEG molecule to the CBS protein. Forexample, PEGylation of htCBS C15S is performed with between about10-fold and 20-fold excess of 5 kDa NHS ester PEG (5NHS), 10 kDa NHSester PEG (10NHS), or 20 kDa NHS ester PEG (20NHS) molecules.

Alternatively, PEGylation is performed with about 40-fold to about60-fold excess of PEG molecule to the CBS protein. In one aspect,PEGylation of htCBS C15S is performed with about 40-fold excess of 5 kDaNHS ester PEG (5NHS), 10 kDa NHS ester PEG (10NHS), or 20 kDa NHS esterPEG (20NHS) molecules. Alternatively, PEGylation of htCBS C15S isperformed with less than about 40-fold excess of 5 kDa NHS ester PEG(5NHS), 10 kDa NHS ester PEG (10NHS), or 20 kDa NHS ester PEG (20NHS)molecules.

In certain embodiments, residual ammonium sulfate from chromatographicpurification is removed from an unmodified batch of CBS protein, such asunmodified htCBS C15S, to increase reproducibility of the PEGylationpattern as compared to the reproducibility of the PEGylation pattern ofhtCBS C15S in a mixture containing the residual ammonium sulfate. Forexample, the ammonium sulfate may be removed using a buffer exchangetechnique.

Following PEGylation, unreacted PEG molecules are removed from thePEGylation mixture to reduce its viscosity by increasing the surfacearea of the diafiltration membrane. For example, the surface area isincreased by about 2-fold, about 3-fold, or about 4-fold. Alternatively,viscosity may be reduced by diluting the PEGylation mixture. Forexample, the PEGylation mixture is diluted by about 2-fold. In certainembodiments, the unreacted PEG molecules or unmodified htCBS C15S isremoved from the mixture to reduce immunogenicity.

III. Treatment with Pegylated htCBS C15S

In certain embodiments, a mammal may be administered htCBS C15SPEGylated with a 5 kDa ester NHS PEG molecule or a 20 kDa ester NHS PEGmolecule. For example, 5NHS PEG-htCBS C15S or 20NHS PEG-htCBS C15S.Administration of 5NHS PEG-htCBS C15S or 20NHS PEG-htCBS C15S reduceslevels of Hcy in plasma or in kidney, liver, or brain tissue by at least40%. For example, Hcy levels are reduced by more than 40%, such as atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 99%. Administration of 5NHS PEG-htCBS C15S or 20NHS PEG-htCBS C15Sincreases levels of Cth in plasma or in kidney, liver, or brain tissueby at least 70%. For example, Cth levels are increased by at least 80%,at least 90%, or at least 99%. Administration of 5NHS PEG-htCBS C15S or20NHS PEG-htCBS C15S increases levels of Cys in plasma or in kidney,liver, or brain tissue by at least 30%. For example, Cys levels areincreased by at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 99%.

Administration of 20NHS PEG-htCBS C15S may reduce levels ofhomolanthionine (Hlth) in plasma or in kidney, liver, or brain tissue byat least 50%. For example, Hlth levels are reduced by at least 60%6, atleast 70%, at least 80%, at least 90%, or at least 99%.

The therapeutic effects of administration of 20NHS PEG-htCBS C15S maycontinue for at least 12 hours of at least 24 hours, at least 48 hours,or at least 72 hours. Specifically, the therapeutic effects may continuefor about 24 hours.

S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) aremetabolites involved in the conversion of methionine to homocysteineduring the proximal portion of the methionine cycle. See, Krijt, et al.J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jul. 15; 877(22-3):2061-2066, which is hereby incorporated by reference herein in itsentirety. In certain embodiments, homocystinuria results in reduction ofthe ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH)(SAM/SAH), which indicates a decrease in methylation capacity. Incertain embodiments, the SAM/SAH ratio may be increased or normalized byadministration of a PEG-htCBS C15S to a subject. For example,administration of a 20NHS PEG-htCBS C15S conjugate reduces SAHaccumulation, thus increasing the SAM/SAH ratio, thereby normalizing thebalance of metabolites in a subject.

IV. Dosing and Administration

In certain embodiments, PEG-htCBS C15S may be administered to a subjectby parenteral administration.

In certain embodiments, PEG-htCBS C15S may be administered to a subjectby subcutaneous (SC), intravenous (IV) or intraperitoneal (IP)injection. As a non-limiting example, PEG-htCBS C15S may be administeredto a subject by subcutaneous administration. As a non-limiting example,PEG-htCBS C15S may be administered to a subject by intravenousadministration. As a non-limiting example, PEG-htCBS C15S may beadministered to a subject by intraperitoneal administration.

In certain embodiments, PEG-htCBS C15S may be administered to a subjectat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more than 20 times.

In certain embodiments, the administration of the PEG-htCBS C15S may berepeated every minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, six6, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes,25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes,55 minutes, hourly, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, daily, 2 days, 3 days, 4 days, 5 days, 6days, weekly, biweekly, 3 weeks, monthly, 2 months, quarterly, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,yearly, 13 months, 14 months, 15 months, 16 months, 17 months, or 18months.

In certain embodiments, the administration of the PEG-htCBS C15S may bea series of doses which are minutes, hours, days or weeks apart. Thenumber of doses in a series may be 2, 3, 4, 5 or 6. As a non-limitingexample, a subject is administered 3 doses 24 hours apart. As anothernon-limiting example, a subject is administered 5 doses 12 hours apart.

In certain embodiments, the administration of the PEG-htCBS C15S mayfollow a dosing schedule of a series of doses that has a gap between thefirst series and the second series of doses. The gap between the dosesmay be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, day, 2 days, 3 days, 4 days, 5 days, 6 days, week, 2 weeks, 3weeks, monthly, 2 months, quarterly, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months,14 months, 15 months, 16 months, 17 months, or 18 months. The number ofdoses in a series may be 2, 3, 4, 5 or 6. As a non-limiting example, asubject may be administered a first series of 5 doses 12 hours apart andthen 14 days after the first dose a subject is administered a secondseries of 5 doses 12 hours apart. As another non-limiting example, asubject is administered two series of doses over a period of 8 weekswhere the first series is one dose twice a week for two weeks and thesecond series of doses is three times a week for 6 weeks.

In one embodiment, PEG-htCBS C15S may be administered at least onceafter a subject has been administered betaine. The time between thebetaine administration the PEG-htCBS C15S may be 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, day, 2 days, 3 days, 4days, 5 days, 6 days, week, 2 weeks, 3 weeks, monthly, 2 months,quarterly, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, or 18 months. As a non-limiting example, thePEG-htCBS C15S may be administered 14 days after the subject wasadministered betaine. As another non-limiting example, a subject may beadministered two doses after the subject was administered betaine. ThePEG-htCBS C15S mutant may be administered 14 and 15 days after betaineadministration.

In one embodiment, PEG-htCBS C15S mutant may be administered incombination with betaine to a subject. The combination may beadministered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or more than 15 times.

In one embodiment, PEG-htCBS C15S mutant may be administered incombination with betaine to a subject after the subject has initiallyreceived betaine. The time between the combination treatment and theoriginal betaine administration may be 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, day, 2 days, 3 days, 4 days, 5days, 6 days, week, 2 weeks, 3 weeks, monthly, 2 months, quarterly, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 13 months, 14 months, 15 months, 16 months, 17months, or 18 months. As a non-limiting example, the combination may beadministered 14 days after the subject was first administered betaine.As another non-limiting example, a subject may be administered two dosesafter the subject was first administered betaine. The combination may beadministered 14 and 15 days after betaine administration.

In one embodiment, the dose of PEG-htCBS C15S mutant administered to asubject may be between 0.01 mg/kg and 1 mg/kg such as 0.05 mg/kg, 0.1mg/kg, 0.5 mg/kg or 1 mg/kg, or between 5 and 8 mg/kg such as 5 mg/kg,5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg or 8 mg/kg.Alternatively, the dose of PEG-htCBS C15S mutant administered to asubject may be between about 2 mg/kg and about 8 mg/kg, about 4 mg/kgand about 16 mg/kg, about 6 mg/kg and 24 mg/kg. For example, the dose isabout 24 mg/kg or about 8 mg/kg.

In one embodiment, the PEG-htCBS C15S may be co-administered withanother therapeutic for treating CBSDH. As used herein,“co-administered” means the administration of two or more components.Components for co-administration include, but are not limited toPEG-htCBS C15S, betaine or Vitamin B6. Co-administration refers to theadministration of two or more components simultaneously or with a timelapse between administration such as 1 second, 5 seconds, 10 seconds, 15seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46minutes, 47 minutes, 48 minutes, 49 minutes, 50 minutes, 51 minutes, 52minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58minutes, 59 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 1.5 days, 2 days,or more than 3 days.

V. Definitions

As used in this specification, the singular forms “a”, “an”, and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to a “polymer” includes a single polymer aswell as two or more of the same or different polymers, or a reference toan “excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

Where a range of values is provided, it is intended that eachintervening value between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure and specifically disclosed. Forexample, if a range of 1 μm to 8 μm is stated, it is intended that 2 μm,3 μm, 4 μm, 5 μm, 6 μm, and 7 μm are also explicitly disclosed, as wellas the range of values greater than or equal to 1 μm and the range ofvalues less than or equal to 8 μm.

“Recombinant,” when used to reference, e.g., a cell, nucleic acid,polypeptide, expression cassette or vector, refers to a material, or amaterial corresponding to the natural or native form of the material,that has been modified by the introduction of a new moiety or alterationof an existing moiety, or is identical thereto but produced or derivedfrom synthetic materials. For example, recombinant cells express genesthat are not found within the native (non-recombinant) form of the cell(i.e., “exogenous nucleic acids”) or express native genes that areotherwise expressed at a different level, typically, under-expressed ornot expressed at all.

Recombinant techniques can include, e.g., use of a recombinant nucleicacid, such as a cDNA encoding a protein or an antisense sequence, forinsertion into an expression system, such as an expression vector; theresultant construct is introduced into a cell, and the cell expressesthe nucleic acid, and the protein, if appropriate. Recombinanttechniques also encompass the ligation of nucleic acids to coding orpromoter sequences from different sources into one expression cassetteor vector for expression of a fusion protein, constitutive expression ofa protein, or inducible expression of a protein.

The terms “subject”, “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, humans.

“Associated” refers to coincidence with the development or manifestationof a disease, condition or phenotype. Association may be due to, but isnot limited to, genes responsible for housekeeping functions whosealteration can provide the foundation for a variety of diseases andconditions, those that are part of a pathway that is involved in aspecific disease, condition or phenotype and those that indirectlycontribute to the manifestation of a disease, condition or phenotype.

“Physiological conditions” or “physiological solution” refers to anaqueous environment having an ionic strength, pH, and temperaturesubstantially similar to conditions in an intact mammalian cell or in atissue space or organ of a living mammal. Typically, physiologicalconditions comprise an aqueous solution having about 150 mM NaCl, pH6.5-7.6, and a temperature of approximately 22-37° C. Generally,physiological conditions are suitable binding conditions forintermolecular association of biological macromolecules. For example,physiological conditions of 150 mM NaCl, pH 7.4, at 37° C. are generallysuitable.

“Pharmaceutically acceptable excipient or carrier” refers to anexcipient that may optionally be included in the compositions of theinvention and that causes no significant adverse toxicological effectsto the patient. The term refers to an excipient that can be taken intothe mammalian subject's body in association with an active compound(e.g. PEG-htCBS C15S) with no significant adverse toxicological effectsto the subject.

The term “excipient” or “vehicle” as used herein means any substance,not itself a therapeutic agent, used as a carrier for delivery of atherapeutic agent and suitable for administration to a subject, e.g. amammal or added to a pharmaceutical composition to improve its handlingor storage properties or to permit or facilitate formation of a doseunit of the composition into a discrete article such as a capsule ortablet suitable for oral administration. Excipients and vehicles includeany such materials known in the art, e.g., any liquid, gel, solvent,liquid diluent, solubilizer, or the like, which is nontoxic, and whichdoes not interact with other components of the composition in adeleterious manner. Administration can mean oral administration,inhalation, enteral administration, feeding or inoculation byintravenous injection. The excipients may include standardpharmaceutical excipients and may also include any components that maybe used to prepare foods and beverages for human and/or animalconsumption, feed or bait formulations or other foodstuffs.

“Permeant”, “drug”, and “pharmacologically active agent” or any othersimilar term means any chemical or biological material or compound,inclusive of peptides, suitable for administration by the methodspreviously known in the art and/or by the methods taught in the presentdisclosure, that induces a desired biological or pharmacological effect,which may include, but is not limited to (1) having a prophylacticeffect on the organism and preventing an undesired biological effectsuch as preventing an infection, (2) alleviating a condition caused by adisease, for example, alleviating pain or inflammation caused as aresult of disease, and/or (3) either alleviating, reducing, orcompletely eliminating the disease from the organism. The effect may belocal, such as providing for a local anesthetic effect, or it may besystemic. This disclosure is not drawn to novel permeants or to newclasses of active agents. Rather it is limited to the mode of deliveryof agents or permeants which exist in the state of the art or which maylater be established as active agents and which are suitable fordelivery by the present disclosure.

The term “about”, particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not be present or occur, so that the descriptionincludes instances where the circumstance is present or occurs andinstances where it is not present or does not occur.

“Substantially absent” or “substantially free” of a certain feature orentity means nearly totally or completely absent the feature or entity.For example, for a subject administered PEG-htCBS C15S, the substantialabsence of an observable side effect means that such side effect iseither non-detectable, or occurs only to a negligible degree, e.g., toan extent or frequency that is reduced by about 50% or more whencompared to either the frequency or intensity of the same side effectobserved in an untreated patient.

The terms “pharmacologically effective amount” or “therapeuticallyeffective amount” as related to the present composition refer to anon-toxic, but sufficient amount of the active agent (or compositioncontaining the active agent) to provide the desired level in thebloodstream or at the site of action (e.g. intracellularly) in thesubject to be treated, and/or to provide a desired physiological,biophysical, biochemical, pharmacological or therapeutic response, suchas amelioration of the manifestations of homocystinuria. The exactamount required will vary from subject to subject, and will depend onnumerous factors, such as the active agent, the activity of thecomposition, the delivery device employed, the physical characteristicsof the composition, intended patient use (i.e., the number of dosesadministered per day), as well as patient considerations, such asspecies, age, and general condition of the subject, the severity of thecondition being treated, additional drugs being taken by the subject,mode of administration, and the like. These factors and considerationscan readily be determined by one skilled in the art, based upon theinformation provided herein. An appropriate “effective” amount in anyindividual case may be determined by one of ordinary skill in the artusing routine experimentation, based upon the information providedherein.

The term “biological activity” refers to any biological activitytypically attributed to a nucleic acid or protein by those skilled inthe art. Examples of biological activities are enzymatic activity,ability to dimerize, fold or bind another protein or nucleic acidmolecule, etc.

The term “nucleic acid” may be in the form of RNA or in the form of DNA,and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA.The DNA may be double-stranded or single-stranded, and ifsingle-stranded may be the coding strand or the non-coding (anti-sense,complementary) strand.

As used herein, a “variant” is a nucleic acid, protein or polypeptidewhich is not identical to, but has significant homology (for example,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%/o sequenceidentity) over the entire length of the wild type nucleic acid or aminoacid sequence, as exemplified by sequences in the public sequencedatabases, such as GenBank. As used herein, a “protein, polypeptide orpeptide fragment thereof” means the full-length protein or a portion ofit having an amino acid sequence usually at least 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acids in length, although dipeptides, tripeptidesand tetrapeptides are also contemplated and encompassed by the presentdisclosure.

As used herein, a “mutant” is a mutated protein designed or engineeredto alter properties or functions relating to glycosylation, proteinstabilization and/or ligand binding.

As used herein, the terms “native” or “wild-type” relative to a givencell, polypeptide, nucleic acid, trait or phenotype, refers to the formin which that is typically found in nature.

As used herein, the terms “protein”, “polypeptide”, “oligopeptide”, and“peptide” have their conventional meaning and are used interchangeablyto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristylation,ubiquitination, etc.). Furthermore, the polypeptides described hereinare not limited to a specific length. Included within this definitionare D- and L-amino acids, and mixtures of D- and L-amino acids. Thisterm also does not refer to or exclude post-expression modifications ofthe polypeptide, for example, glycosylations, acetylations,phosphorylations and the like, as well as other modifications known inthe art, both naturally occurring and non-naturally occurring. Apolypeptide may be an entire protein, or a subsequence thereof.Polypeptides can also refer to amino acid subsequences comprisingepitopes, i.e., antigenic determinants substantially responsible for theimmunogenic properties of a polypeptide and being capable of evoking animmune response.

“Position corresponding to” refers to a position of interest (i.e., basenumber or residue number) in a nucleic acid molecule or protein relativeto the position in another reference nucleic acid molecule or protein.Corresponding positions can be determined by comparing and aligningsequences to maximize the number of matching nucleotides or residues,for example, such that identity between the sequences is greater than90%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99%. The position of interest is then given thenumber assigned in the reference nucleic acid molecule. For example, ifa particular polymorphism in Gene-X occurs at nucleotide 2073 of SEQ IDNo. X, to identify the corresponding nucleotide in another allele orisolate, the sequences are aligned and then the position that lines upwith 2073 is identified. Since various alleles may be of differentlength, the position designated 2073 may not be nucleotide 2073, butinstead is at a position that “corresponds” to the position in thereference sequence.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage may be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package),or by visual inspection (see generally, Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1995 Supplement) (Ausubel)). Examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity arethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length “W” in the query sequence, which either match or satisfysome positive-valued threshold score “T” when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as “seeds” for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

While all the above-mentioned algorithms and programs are suitable for adetermination of sequence alignment and % sequence identity, forpurposes of the disclosure herein, determination of % sequence identitywill typically be performed using the BESTFIT or GAP programs in the GCGWisconsin Software package (ACCELRYS®, Madison Wis.), using defaultparameters provided.

The term “L or D form amino acids” may be used in relation to thepeptide described herein. As will be recognized by skilled artisans, thevarious X^(n) residues comprising the compounds of the invention may bein either the L- or D-configuration about their C_(α) carbons. In oneembodiment, all the C_(α) carbons of a particular compound are in thesame configuration. In some embodiments of the invention, the compoundscomprise specific chiralities about one or more C_(α) carbon(s) and/orinclude non-peptide linkages at specified locations to impart thecompound with specified properties. For example, it is well-known thatpeptides composed in whole or in part of D-amino acids are moreresistant to proteases than their corresponding L-peptide counterparts.Thus, in one embodiment, the compounds are peptides composed in whole orin part of D-amino acids. Alternatively, compounds having good stabilityagainst proteases may include peptide analogs including peptide linkagesof reversed polarity at specified positions. For example, compoundshaving stability against tryptic-like proteases include peptide analogshaving peptide linkages of reversed polarity before each L-Arg or L-Lysresidue; compounds having stability against chymotrypsin-like proteasesinclude peptide analogs having peptide linkages of reversed polaritybefore each small and medium-sized L-aliphatic residue or L-non-polarresidue. In another embodiment, compounds having stability againstproteases include peptide analogs composed wholly of peptide bonds ofreversed polarity. Other embodiments having stability against proteaseswill be apparent to those of skill in the art. Additional specificembodiments of the compounds are described herein.

Described herein are compositions, methods, processes, kits and devicesfor the design, preparation, manufacture and/or formulation of PEG-htCBSC15S proteins. The details of one or more embodiments of the inventionare set forth in the accompanying description below. Although anymaterials and methods similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred materials and methods are now described. Other features,objects and advantages of the invention will be apparent from thedescription. In the description, the singular forms also include theplural unless the context clearly dictates otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. In the case of conflict, the presentdescription will control.

The present invention is further illustrated by the followingnon-limiting examples.

VI. Examples Example 1. Experimental Procedures A. Chemicals

Unless stated otherwise, all materials were purchased fromSIGMA-ALDRICH® or FISHER SCIENTIFIC™. L-[¹⁴C]-serine was obtained fromPERKIN ELMER® Life Sciences.

B. Animals

All animal procedures were approved under animal protocol #B-49414(03)1Eby the University of Colorado Denver Institutional Animal Care and UseCommittee (IACUC), which is an AAALAC INTERNATIONAL®-accredited(#00235), Public Health Service-assured (#A 3269-01) and USDA-licensed(#84-R-0059) institution. “Human Only” (HO) mice were previouslygenerated and propagated and genotyped as described in Maclean et al.,Mol. Genet. Metab. 2010, 101, (2-3), 153-62; Bublil et al., J ClinInvest 2016, 126, (6), 2372-84; and International applicationPCT/2016/061050, which are hereby incorporated by reference in theirentireties. Animals were maintained on extruded standard diet 2918,2919, or 2920X from TEKLAD GLOBAL RODENT DIETS® (Harlan, Livermore,Calif., USA). A single-use lancet for submandibular bleeding was usedfor blood collection into CAPIJECT® T-MLHG lithium heparin (12.5 IU)tubes with gel (Terumo Medical Corporation, NJ, USA). Tubes were thencentrifuged at 1,200×g for 10 minutes, followed by transfer of plasma to1.5 ml tubes and storage at −80° C.

C. Determination of Metabolite Concentrations

Plasma metabolites were determined by stable-isotope-dilution gaschromatography mass spectrometry as previously described. See, Allen etal., Metabolism 1993, 42, 1448-1460. The analysis was performed in ablinded fashion without knowing the animal genotype and/or treatmentregimen.

D. Size Exclusion Chromatography-High Performance Liquid Chromatography(SEC-HPLC)

CBS and PEG-htCBS C15S preparations were separated on BioSEC-5 7.8×300mm, 1000 Å pore size column (Agilent, CA, USA). Column was equilibratedand operated in 100 mM sodium phosphate, pH 6.8, 150 mM NaCl at a flowrate of 1 ml/min at room temperature. Protein samples diluted in mobilephase to a final concentration of 2 mg/ml were loaded on a column via 10μl sample loop and detected using UV detector at wavelength of 225 nm.Chromatograms were recorded and analyzed using 32 karat software(BECKMAN COULTER®, CA, USA).

E. Non-Reduced Capillary Electrophoresis (NR-CE)

CBS and PEG-htCBS C15S preparations were analyzed on PA800 capillaryelectrophoresis system (BECKMAN COULTER®, CA, USA) equipped with 30 cmlong, 50 μm ID bare-fused silica capillary. The capillary was cut toprovide a total length of 30 cm with a distance of approximately 20 cmfrom sample introduction inlet to detection window and installed in acartridge with a 100×800 μm aperture. The capillary was preconditionedwith 0.1 M NaOH wash followed by 0.1 M HCl wash before rinsing it withwater and final equilibration with SDS MW sample buffer. Protein samplesof concentrations less than 10 mg/ml and in a formulation containinghigh salt were buffer exchanged in water twice (about 7-fold dilutioneach time) to remove matrix interference, diluted to 1 mg/ml finalconcentration, and denatured by heating at 80° C. for 5 minutes. Thesample was loaded by a high-resolution method using electrokineticinjection (5 kV) for a duration of 20 seconds and separated for 50minutes under voltage of 15 kV. Electropherograms were recorded at a UVwavelength of 220 nm and analyzed using 32 karat software (BECKMANCOULTER®, CA, USA).

F. Protein Purification and PEGylation

Human truncated CBS carrying the C15S mutation (htCBS C15S) wasexpressed and purified as described in Bublil et al., J Clin Invest2016, 126, (6), 2372-84, International application PCT/2016/061050,which are hereby incorporated by reference in their entireties. Thepurified enzyme was formulated and concentrated using the LABSCALE™Tangential Flow Filter (TFF) system (EMD Millipore, MA, USA) equippedwith PELLICON® XL 50 ULTRAcel 30 cartridge (regenerated cellulose 30 kDamolecular weight cut-off (MWCO) membrane) into 200 mM potassiumphosphate pH 6.5 or 100 mM sodium phosphate pH 7.2. Activated PEGmolecules with either a maleimide or a NHS ester coupling group werepurchased from NOF Corporation (Japan). PEGylation of htCBS C15S withmaleimide and NHS ester PEG molecules was carried out in 100 mMpotassium phosphate pH 6.5 and 50 mM sodium phosphate pH 7.2,respectively, by adding PEG molecules dissolved in MILLIQ® purifiedwater in the desired molar ratio (typically CBS protein:PEG=1:10) to afinal protein concentration of 5 mg/ml. Reaction was carried out at 4°C. overnight. Subsequently, reaction mixture was diluted twice withMILLIQ® purified water, buffer exchanged into Gibco 1×PBS (Thermo FisherScientific, MA, USA) and concentrated using LABSCALE™ TFF system withPELLICON® XL 50 Biomax 100 cartridge (polyether sulfone 100 kDa MWCOmembrane). The final PEG-htCBS C15S conjugates were filter sterilized(Millipore's 0.2 μm PVDF membrane filter), aliquoted, flash frozen inliquid nitrogen and stored at −80° C.

G. Protein Gel Electrophoresis

Protein concentrations were determined by the Bradford assay (ThermoPierce, MA, USA) using bovine serum albumin (BSA) as a standard per themanufacturer's recommendations. Denatured proteins were separated bySDS-PAGE using a 10% Mini-PROTEAN® Tris-glycine extended (TGX) gels(BIORAD®, CA, USA), while native samples were resolved by native PAGE in4-15% Mini-PROTEAN® TGX gels. For visualization, the gels were stainedwith Simple Blue (Invitrogen, CA, USA). Free PEG molecules and PEGylatedhtCBS C15S were visualized using barium iodide staining as described inKurfurst et al., Anal Biochem 1992, 200, (2), 244-8, which is herebyincorporated by reference in its entirety.

H. CBS Activity Assays

The CBS activity was determined by a previously described radioisotopeassay using L-[¹⁴C]-serine as the labeled substrate with the followingmodification. See, Kraus et al., Methods Enzymol 1987, 143, 388-94;Bublil et al., J Clin Invest 2016, 126, (6), 2372-84, each of which arehereby incorporated by reference in their entireties. Four or five μl ofundiluted plasma was assayed in a total volume of 100 μl for 30 minutes.The reaction was terminated by mixing of 20 μl of a reaction mixturewith 5 μL of chilled performic acid (30% H₂O₂:100% formic acid=1:9) andfurther processed as described in the original protocol.

I. PEGylation Mapping

Mapping of maleimide and NHS ester PEGylation of htCBS C15S wasperformed using ESI-QTOF mass spectrometer model 6538 (Agilent, CA,USA). Unmodified enzyme as a control and PEGylated CBS were used as is(unmodified htCBS C15S, 20NHS PEG htCBS-C15S, and 400MA PEG-htCBS C15S)or dePEGylated by incubating the sample at pH 11.5 for 22 hours at 37°C. (20NHS PEG-htCBS C15S). All samples were buffer exchanged, denatured,reduced with 10 mM DTT and alkylated with 30 mM iodoacetamide priordigestion. The 400MA PEG-htCBS C15S and 20NHS PEG-htCBS C15S weredigested overnight with Lys-C and Asp-N (1:50 (w/w) ratio, 37° C.),respectively. The 20NHS PEG-htCBS C15S conjugates were also chemicallydigested using 2-nitro-5-thiocyanobenzoic acid (NTCB) to furtherdifferentiate which cysteine residues had been modified. Peptides wereseparated on a C18 column (Xbridge BEH130 3.5 μm, 4.6×150 mm) usingRP-HPLC with UV detection (Agilent), and LC/UV/MS/MS chromatograms wereanalyzed using MassHunter software (Agilent, CA, USA). Peptide mappingand sequence matching was performed using BioConfirm package (Agilent,CA, USA) with manual validation to ensure correct assignment.

J. Statistical Analysis

All data are presented as mean f standard error of the mean (SEM).Statistical comparisons of 2 groups were conducted using an unpaired,two tailed Students t-test. Statistical analysis of 3 or more factorlevels was conducted by ANOVA followed by Tukey's multiple comparisontest to determine significance. The p value of less than 0.05 wasdetermined to correspond to statistical significance.

Example 2. Optimizing PEGylation of htCBS C15S

To understand the PEGylation patterns and to optimize the conjugationreaction to yield a more uniform modification, the individual PEG-htCBSC15S species were separated and the residues targeted with the ME-400MAmaleimide PEG molecules were later identified (FIG. 2 ). First, thePEGylation reaction was loaded onto a DEAE Sepharose column equilibratedin a buffer with low conductivity and higher pH (10 mM TRIS.HCl pH 8.5)to enhance binding of all 400MA PEG-htCBS C15S species and ran on ashallow salt gradient (5%, 10-110 mM NaCl) to elute the protein. FIG. 2shows a chromatogram of such a run and the recovery of two pools, whichwere subsequently analyzed along with an unmodified htCBS C15S and 400MAPEG-htCBS C15S reaction mixture on SDS-PAGE and native PAGE. Gelanalysis of the pools provided evidence that the 400MA PEG-htCBS C15Sconsisted of at least 3 different PEG-htCBS C15S species.

Pool 1 (P1) was enriched in forms which had a weaker negative charge(FIG. 2 ) and more PEG moieties attached than those recovered in pool 2(P2). The predominant 400MA PEG-htCBS C15S conjugate in P1 consisted ofa dimeric htCBS C15S, where each protein was modified with ME-400MA tothe same extent, most likely 2 PEGs per CBS dimer (homoPEGylated dimer).Moreover, P1 contained a minor 400MA PEG-htCBS C15S species with anadditional 1 or 2 PEG moieties. On the other hand, the major 400MAPEG-htCBS C15S form of P2 contained a dimeric enzyme, where just one ofthe proteins was modified with PEG, i.e. 1 PEG per CBS dimer(hemiPEGylated dimer). Separation of individual 400MA PEG-htCBS C15Sspecies enabled the identification of the sites of maleimide PEGylationusing LC-MS.

For phase I, the unmodified htCBS C15S, P1 and P2 were digested withLys-C. Results of LC-MS are shown in FIG. 3A, FIG. 3B, and FIG. 3C. Outof eight cysteine-containing peptides identified from thereduced/alkylated LC-MS peptide map, seven such peptides yielded similarrelative abundances in all three samples indicating that these peptideswere unlikely to have been PEGylated. Only one peptide (272-322; SEQ IDNO. 31) containing two cysteine residues (C272 and C275) showed asignificant decrease in each of the two 400MA PEG-htCBS C15S species (P1and P2).

To further differentiate whether C272 and/or C275 were modified in phaseII, the two residues would need to be separated into different peptides.Reagent NTCB cleaves N-terminally to a free cysteine residue. Results ofLC-MS of an unmodified reference, homoPEGylated, and hemiPEGylatedproteins are shown in FIG. 4A, FIG. 4B, and FIG. 4C.

Only one peptide (244-271; SEQ ID NO: 32) showed a significant decreasein 400MA PEG-htCBS C15S forms (more in P1 than for P2) compared tosimilarly treated unmodified htCBS C15S. The yield for the 244-271peptide was lowered in the homoPEGylated 400MA PEG-htCBS C15S P1 sampleand to a lesser extent in the hemiPEGylated 400MA PEG-htCBS C15S P2.

The peptide recovery signifies that C272 is blocked, thus the loweryield of the 244-271 peptide in 400MA PEG-htCBS C15S forms signifiesthat C272 is the primary site for maleimide PEGylation. It is possiblethat both cysteine residues C272 and C275 are involved in the PEGylationof higher molecular weight species present in the low abundance in P1.Low NTCB digestion efficiency resulted in low abundance of the recoveredpeptides.

Example 3. Reproducibility of PEGylation of htCBS C15S with NHS EsterPEG Molecules

The issues with reproducibility of htCBS C15S PEGylation with maleimidePEGs led to analysis of other PEGylation chemistries, particularly theuse of NHS ester-activated PEGs. First, modification of htCBS C15S wasassessed with three NHS ester PEG molecules to determine their impact onenzyme activity. Four, three and two different PEG:CBS molar ratios weretested in PEGylations with 5, 10 and 20 kDa linear NHS ester PEGmolecules, respectively. Unlike the site-selective maleimide PEGylation,the NHS ester PEG molecules yielded a reproducible mixture of highlymodified isomers that were practically inseparable using SDS-PAGE aswell as by native PAGE or SEC-HPLC.

For example, htCBS C15S modified by three linear 5, 10, and 20 kDa NHSester PEG molecules (5NHS, 10NHS, and 20NHS) in different molar excessesof PEG molecules to the CBS protein were separated on SDS-PAGE (BIORAD®10% Mini-PROTEAN® TGX gel) or native PAGE (BIORAD® 4-15% Mini-PROTEAN®TGX gel) and stained with Safe Stain (Invitrogen) per manufacturer'srecommendation. Partial separation was achieved only using NR-CE (FIG. 1), which served as a method for “fingerprinting” of the 20NHS PEG-htCBSC15S production batches.

While a 10-20-fold molar excess of PEG molecules to the CBS proteinyielded NHS PEG-htCBS C15S species with only a slight effect on CBSactivity, higher excess of PEG, such as 40- and 60-fold for 5 kDa and40-fold for 10 kDa NHS PEG, resulted in a markedly reduced activity ofthe respective NHS PEG-htCBS C15S conjugates (FIG. 5 ). Thus,conjugation of htCBS C15S with either NHS PEG molecules in a PEGylationreaction with no more than 20-fold PEG:CBS molar excess yielded highlymodified, bulky NHS PEG-htCBS C15S conjugates with essentially fullyretained activity. PEGylation of htCBS C15S with lysine-targeting 5, 10or 20 kDa NHS ester PEGs used in up to a 20-fold molar excess to htCBSC15S protein did not significantly affect the enzyme's specific activity(FIG. 5 ), a property shared with the maleimide PEGs (maleimidePEGylation data shown in Bublil et al., J Clin Invest 2016, 126, (6),2372-84 and International application PCT/2016/061050, which are herebyincorporated by reference in their entireties).

PEGylation of htCBS C15S with 20NHS ester PEG molecules showedreproducibility. Similarly, as done for the maleimide PEGs, thereproducibility of htCBS C15S PEGylation with 20NHS PEG (10-fold PEG:CBSmolar excess) was assessed. Initial batches had a highly variable extentof PEGylation (data not shown). However, the main culprit of variabilitywas identified as the residual ammonium sulfate in unmodified htCBSC15S. An extensive buffer exchange scheme was employed at the end of thechromatographic purification (15-20 diavolumes) to formulate the enzymeinto the pre-PEGylation buffer, which resulted in a reproduciblePEGylating pattern.

Four batches of purified unmodified htCBS C15S were PEGylated with 20NHSPEG molecules yielding four 20NHS PEG-htCBS C15S conjugates. Therespective 4 pairs of unmodified enzyme (pre) and 20NHS PEG-htCBS C15Sconjugate (post) were designated as LAB, 10L, TR1 and TR2. The proteinsamples were resolved in SDS-PAGE (BIORAD® 10% Mini-PROTEAN® TGX gel) ornative PAGE (BIORAD® 4-15% Mini-PROTEAN® TGX gel) and stained with SafeStain (Invitrogen) per manufacturer's recommendation or iodine stainedto detect PEG. Unmodified htCBS C15S and free unreacted PEG moleculeswere also observed.

Compared to all the previous PEG-htCBS C15S conjugates, the 20NHSPEG-htCBS C15S showed increased viscosity (data not shown) and thusthere was a concern of sufficient free, unreacted PEG molecule removalduring the final (post PEGylation) buffer exchange. Indeed, processingof the initial batches (for example LAB and 10L) yielded a large amountof unreacted PEG molecules carried over to the final product. Correctivemeasures, which included removal of 20% DMSO from the mixture, higherMWCO membrane cartridge for TFF unit (100 versus 30 kDa), 3-fold largersurface area and 2-fold dilution of the PEGylation mixture prior bufferexchange, all helped to reduce the viscosity and assisted in betterclearance of free PEG molecules during the final formulation (TR1 andTR2).

Example 4. Characterizing the Extent of PEGylation Using NHS Ester PEGMolecules

SDS and native PAGE were not observed to sufficiently resolve individual20NHS PEG-htCBS C15S species. Two additional methods were developed forcharacterization of the extent to which the htCBS C15S was modified with20NHS PEG molecules allowing for a quick assessment of PEGylation inproduction and providing a definition of acceptance criteria for theproduct (FIG. 1 ).

One method relied on comparison of SEC-HPLC chromatographic profiles ofindividual 20NHS PEG-htCBS C15S batches compared with that of unmodifiedhtCBS C15S as shown in Table 1.

TABLE 1 UV 225 nm Absorbance [AU] values for different batches Batchidentifier un- modified Time htCBS [min] C15S 6-70 6-89 8-14 8-15 8-168-22 8-23 8-24 8-25 0.00 2.30E−05 2.00E−06 1.00E−05 1.00E−06 1.30E−058.00E−06 −1.20E−05 −3.30E−05 −1.00E−06 1.00E−05 0.02 2.70E−05 4.00E−065.00E−06 2.00E−06 4.00E−06 −1.20E−05 1.00E−06 −1.50E−05 −3.20E−056.00E−06 0.03 2.10E−05 −1.00E−06 1.80E−05 6.00E−06 1.80E−05 5.00E−06−1.60E−05 −2.30E−05 −1.30E−05 1.50E−05 0.05 1.40E−05 −6.00E−06 1.50E−054.00E−06 5.00E−06 5.00E−06 −6.00E−06 −1.50E−05 −2.40E−05 6.00E−06 0.071.20E−05 1.60E−05 3.30E−05 8.00E−06 5.00E−06 8.00E−06 3.00E−06 −2.10E−05−1.80E−05 6.00E−06 0.08 1.20E−05 1.20E−05 7.00E−06 4.00E−06 −5.00E−061.40E−05 6.00E−06 6.00E−06 −7.00E−06 4.00E−06 0.10 1.30E−05 −8.00E−061.70E−05 1.20E−05 7.00E−06 −1.00E−06 1.00E−06 −1.50E−05 −2.30E−056.00E−06 0.12 3.10E−05 1.30E−05 4.00E−06 1.00E−06 1.50E−05 8.00E−062.00E−06 5.00E−06 −1.30E−05 1.00E−05 0.13 2.70E−05 4.00E−06 1.00E−052.70E−05 1.40E−05 1.80E−05 1.20E−05 −6.00E−06 −2.50E−05 1.10E−05 0.152.20E−05 1.00E−06 2.40E−05 1.30E−05 −1.00E−06 2.30E−05 2.00E−06 0.00E+00−1.80E−05 7.00E−06 0.17 1.60E−05 1.40E−05 1.20E−05 2.20E−05 1.20E−051.00E−06 −3.00E−06 5.00E−06 −1.20E−05 1.80E−05 0.18 1.10E−05 −4.00E−061.30E−05 1.00E−05 −1.00E−06 −3.00E−06 3.00E−06 6.00E−06 −1.80E−051.50E−05 0.20 1.40E−05 2.00E−06 1.50E−05 3.00E−06 1.60E−05 1.60E−05−8.00E−06 1.20E−05 −2.30E−05 1.80E−05 0.22 2.10E−05 1.30E−05 1.00E−053.40E−05 1.00E−05 7.00E−06 0.00E+00 5.00E−06 −2.70E−05 1.60E−05 0.231.70E−05 5.00E−06 8.00E−06 1.00E−06 1.00E−06 1.70E−05 1.10E−05 1.00E−06−2.30E−05 1.80E−05 0.25 1.70E−05 1.20E−05 2.50E−05 1.00E−05 6.00E−065.00E−06 −1.30E−05 −7.00E−06 −3.20E−05 2.50E−05 0.27 8.00E−06 −1.00E−062.70E−05 1.20E−05 1.40E−05 1.30E−05 −1.20E−05 −4.00E−06 −2.70E−052.40E−05 0.28 2.20E−05 2.00E−06 8.00E−06 2.10E−05 −3.00E−06 2.00E−067.00E−06 1.30E−05 −2.80E−05 3.10E−05 0.30 2.60E−05 −3.00E−06 1.80E−052.60E−05 5.00E−06 1.40E−05 −6.00E−06 7.00E−06 −3.10E−05 2.00E−05 0.321.70E−05 7.00E−06 2.40E−05 −4.00E−06 5.00E−06 1.00E−06 2.00E−06−7.00E−06 −3.10E−05 1.50E−05 0.33 1.40E−05 5.00E−06 2.20E−05 3.10E−051.30E−05 1.00E−05 2.00E−06 8.00E−06 −3.00E−05 1.50E−05 0.35 1.50E−056.00E−06 1.10E−05 2.00E−06 6.00E−06 6.00E−06 −5.00E−06 7.00E−06−1.80E−05 2.30E−05 0.37 8.00E−06 1.00E−06 4.00E−06 1.80E−05 0.00E+002.80E−05 −3.00E−06 4.00E−06 −3.80E−05 2.40E−05 0.38 2.00E−06 0.00E+004.40E−05 2.70E−05 1.00E−05 1.60E−05 2.00E−06 3.00E−06 −3.40E−05 3.10E−050.40 1.00E−05 1.30E−05 4.00E−05 7.00E−06 4.00E−06 1.00E−06 1.10E−051.70E−05 −3.50E−05 3.40E−05 0.42 1.40E−05 −3.00E−06 1.80E−05 4.00E−065.00E−06 2.00E−05 1.00E−05 0.00E+00 −3.40E−05 3.40E−05 0.43 6.00E−06−4.00E−06 2.20E−05 1.00E−05 1.60E−05 2.10E−05 5.00E−06 −3.00E−06−3.80E−05 2.70E−05 0.45 1.00E−05 −1.00E−05 1.80E−05 1.20E−05 3.30E−051.10E−05 1.10E−05 −8.00E−06 −2.60E−05 3.30E−05 0.47 6.00E−06 −1.10E−052.00E−05 2.00E−06 0.00E+00 1.10E−05 2.40E−05 −1.20E−05 −2.80E−052.50E−05 0.48 1.20E−05 1.10E−05 2.00E−05 1.60E−05 2.20E−05 1.80E−056.00E−06 −4.00E−06 −3.00E−05 3.70E−05 0.50 6.00E−06 2.00E−06 2.10E−051.60E−05 4.00E−06 1.20E−05 3.20E−05 −7.00E−06 −3.30E−05 3.10E−05 0.523.20E−05 1.20E−05 1.70E−05 4.00E−06 0.00E+00 6.00E−06 1.00E−05 −1.10E−05−3.50E−05 2.40E−05 0.53 1.50E−05 7.00E−06 2.50E−05 2.30E−05 4.00E−060.00E+00 2.30E−05 0.00E+00 −3.30E−05 2.40E−05 0.55 1.10E−05 7.00E−062.80E−05 2.60E−05 4.00E−06 1.60E−05 3.60E−05 −2.00E−05 −4.00E−052.70E−05 0.57 8.00E−06 −4.00E−06 5.20E−05 1.00E−05 3.00E−06 8.00E−061.50E−05 −2.00E−05 −2.10E−05 5.40E−05 0.58 1.50E−05 1.20E−05 3.00E−056.00E−06 1.20E−05 5.00E−06 1.80E−05 −1.60E−05 −2.50E−05 2.30E−05 0.601.20E−05 1.00E−05 4.50E−05 5.00E−06 −2.00E−06 2.00E−05 2.30E−05−1.80E−05 −1.30E−05 2.00E−05 0.62 2.00E−06 2.70E−05 3.50E−05 3.20E−057.00E−06 2.00E−06 3.10E−05 −1.80E−05 −3.20E−05 3.10E−05 0.63 1.40E−05−8.00E−06 4.30E−05 1.10E−05 1.40E−05 1.30E−05 2.80E−05 −2.80E−05−3.80E−05 3.00E−05 0.65 7.00E−06 −7.00E−06 3.80E−05 1.30E−05 3.00E−061.40E−05 2.60E−05 −2.10E−05 −3.10E−05 2.70E−05 0.67 1.00E−06 −1.60E−054.20E−05 2.80E−05 −1.00E−06 8.00E−06 2.10E−05 −2.70E−05 −1.70E−054.10E−05 0.68 2.30E−05 0.00E+00 3.80E−05 2.20E−05 4.00E−06 1.60E−052.40E−05 −2.10E−05 −2.30E−05 3.10E−05 0.70 1.50E−05 −1.00E−06 3.30E−052.60E−05 3.00E−06 1.20E−05 2.80E−05 −3.10E−05 −3.40E−05 2.70E−05 0.72−1.00E−06 −4.00E−06 2.00E−05 1.50E−05 1.10E−05 3.50E−05 1.50E−05−2.60E−05 −3.20E−05 3.80E−05 0.73 7.00E−06 3.00E−06 4.00E−05 1.60E−051.40E−05 1.10E−05 3.20E−05 −2.30E−05 −2.30E−05 4.50E−05 0.75 1.80E−054.00E−06 4.50E−05 3.00E−05 1.30E−05 1.40E−05 2.80E−05 −3.30E−05−1.50E−05 3.20E−05 0.77 1.60E−05 3.00E−06 3.40E−05 1.30E−05 1.00E−057.00E−06 4.00E−05 −2.00E−05 −3.80E−05 2.70E−05 0.78 3.10E−05 −1.20E−053.60E−05 1.10E−05 5.00E−06 5.00E−06 3.70E−05 −3.20E−05 −2.80E−053.40E−05 0.80 5.00E−06 −1.00E−05 3.60E−05 1.80E−05 6.00E−06 1.30E−052.40E−05 −2.20E−05 −2.10E−05 3.00E−05 0.82 8.00E−06 −1.00E−05 3.10E−052.20E−05 1.00E−05 7.00E−06 2.70E−05 −2.10E−05 −3.10E−05 4.30E−05 0.833.80E−05 −1.00E−06 3.70E−05 2.30E−05 3.00E−06 1.10E−05 3.40E−05−3.00E−05 −3.50E−05 2.40E−05 0.85 1.60E−05 2.00E−06 5.10E−05 2.20E−051.50E−05 2.20E−05 2.50E−05 −2.80E−05 −2.80E−05 4.00E−05 0.87 4.00E−061.00E−06 3.60E−05 1.40E−05 3.00E−06 1.40E−05 3.40E−05 −3.70E−05−2.30E−05 4.80E−05 0.88 3.50E−05 7.00E−06 4.60E−05 3.80E−05 1.70E−051.20E−05 3.40E−05 −3.00E−05 −1.40E−05 3.70E−05 0.90 3.10E−05 6.00E−064.70E−05 2.60E−05 1.70E−05 5.00E−06 3.40E−05 −3.60E−05 −2.10E−053.30E−05 0.92 1.80E−05 −1.10E−05 5.10E−05 2.20E−05 1.20E−05 −2.00E−063.30E−05 −2.50E−05 −1.40E−05 3.40E−05 0.93 2.20E−05 −7.00E−06 4.20E−052.00E−05 2.00E−05 1.60E−05 3.80E−05 −2.50E−05 −1.20E−05 7.50E−05 0.951.10E−05 2.00E−06 3.50E−05 2.10E−05 1.40E−05 4.00E−06 3.20E−05 −2.80E−05−1.20E−05 4.20E−05 0.97 1.70E−05 4.00E−06 3.60E−05 4.60E−05 −3.00E−06−1.00E−06 3.40E−05 −4.40E−05 −1.50E−05 4.20E−05 0.98 1.50E−05 1.20E−055.50E−05 3.30E−05 1.60E−05 2.50E−05 3.80E−05 −3.60E−05 −1.00E−064.50E−05 1.00 1.30E−05 7.00E−06 5.10E−05 3.50E−05 1.60E−05 3.60E−054.00E−05 −3.80E−05 −2.00E−05 3.80E−05 1.02 6.00E−06 −1.00E−06 3.40E−053.00E−05 6.00E−06 7.00E−06 3.40E−05 −5.00E−06 −1.50E−05 2.60E−05 1.031.30E−05 2.00E−06 3.70E−05 5.20E−05 1.30E−05 2.00E−05 3.50E−05 −4.30E−05−3.00E−06 2.10E−05 1.05 1.60E−05 −5.00E−06 4.60E−05 2.60E−05 1.20E−051.40E−05 5.40E−05 −3.40E−05 −2.10E−05 4.10E−05 1.07 2.20E−05 −1.00E−064.20E−05 3.10E−05 1.30E−05 8.00E−06 4.50E−05 −3.60E−05 −2.40E−054.40E−05 1.08 3.20E−05 −1.00E−05 3.50E−05 4.50E−05 1.20E−05 4.00E−064.60E−05 −4.30E−05 −1.80E−05 3.70E−05 1.10 2.10E−05 −1.10E−05 2.80E−053.00E−05 2.60E−05 8.00E−06 4.10E−05 −4.00E−05 −1.50E−05 2.80E−05 1.122.70E−05 2.00E−06 5.40E−05 2.40E−05 3.00E−06 1.10E−05 6.10E−05 −3.20E−05−2.50E−05 3.60E−05 1.13 8.00E−06 −1.10E−05 4.30E−05 3.60E−05 6.00E−061.20E−05 4.10E−05 −3.60E−05 −2.10E−05 2.30E−05 1.15 3.00E−05 −1.20E−055.40E−05 2.70E−05 2.50E−05 1.80E−05 4.00E−05 −4.20E−05 −3.50E−052.80E−05 1.17 2.60E−05 3.00E−06 6.30E−05 2.20E−05 2.60E−05 1.40E−054.20E−05 −3.60E−05 −2.70E−05 3.40E−05 1.18 3.00E−05 8.00E−06 3.70E−052.30E−05 1.40E−05 1.50E−05 5.10E−05 −4.50E−05 −3.60E−05 4.20E−05 1.202.60E−05 −2.00E−06 4.40E−05 2.10E−05 2.00E−05 2.00E−05 4.10E−05−4.10E−05 −2.50E−05 3.00E−05 1.22 1.70E−05 1.00E−06 5.50E−05 3.10E−056.00E−06 5.00E−06 4.00E−05 −2.30E−05 −2.20E−05 3.40E−05 1.23 1.60E−057.00E−06 6.20E−05 2.60E−05 3.10E−05 4.00E−06 6.30E−05 −3.80E−05−2.40E−05 2.80E−05 1.25 −1.00E−06 −1.10E−05 5.10E−05 3.10E−05 3.00E−061.60E−05 5.50E−05 −3.80E−05 −2.50E−05 3.10E−05 1.27 5.00E−06 −1.00E−065.00E−05 3.00E−05 1.50E−05 2.20E−05 6.10E−05 −3.40E−05 −1.70E−052.60E−05 1.28 1.10E−05 −1.10E−05 4.20E−05 1.20E−05 1.60E−05 1.40E−056.40E−05 −3.60E−05 −2.20E−05 3.00E−05 1.30 1.80E−05 −3.00E−06 5.70E−052.10E−05 1.40E−05 1.20E−05 5.20E−05 −3.10E−05 −1.10E−05 3.00E−05 1.322.70E−05 −1.00E−05 5.00E−05 2.10E−05 2.20E−05 1.50E−05 4.60E−05−2.00E−05 −3.00E−05 3.40E−05 1.33 2.50E−05 −7.00E−06 4.30E−05 3.10E−052.00E−05 1.20E−05 6.50E−05 −3.40E−05 −3.40E−05 2.10E−05 1.35 1.20E−05−1.00E−06 5.40E−05 2.30E−05 2.80E−05 1.80E−05 6.40E−05 −4.30E−05−2.00E−05 2.30E−05 1.37 1.60E−05 2.00E−06 5.00E−05 2.30E−05 4.30E−050.00E+00 7.50E−05 −4.20E−05 −1.40E−05 2.40E−05 1.38 1.60E−05 −3.00E−064.70E−05 1.40E−05 1.00E−05 1.20E−05 7.30E−05 −3.50E−05 −2.00E−052.70E−05 1.40 2.80E−05 5.00E−06 5.00E−05 2.40E−05 1.70E−05 3.30E−057.00E−05 −3.10E−05 −1.60E−05 4.00E−05 1.42 8.00E−06 3.00E−06 5.20E−051.20E−05 2.30E−05 2.00E−05 7.10E−05 −4.50E−05 −2.30E−05 3.50E−05 1.432.60E−05 6.00E−06 6.30E−05 3.00E−06 2.40E−05 2.80E−05 6.50E−05 −4.10E−05−1.80E−05 2.40E−05 1.45 3.70E−05 −4.00E−06 4.60E−05 2.60E−05 2.20E−052.30E−05 7.20E−05 −1.80E−05 −3.30E−05 2.60E−05 1.47 1.70E−05 −1.10E−055.50E−05 1.70E−05 2.50E−05 1.10E−05 6.30E−05 −3.00E−05 −2.10E−052.80E−05 1.48 1.80E−05 2.10E−05 6.00E−05 2.60E−05 2.80E−05 3.70E−056.60E−05 −4.30E−05 −1.30E−05 1.20E−05 1.50 3.50E−05 0.00E+00 5.30E−052.30E−05 2.70E−05 2.40E−05 5.40E−05 −3.30E−05 −8.00E−06 3.50E−05 1.522.80E−05 1.10E−05 6.10E−05 1.70E−05 2.20E−05 4.50E−05 7.30E−05 −3.40E−05−3.10E−05 3.20E−05 1.53 2.80E−05 1.00E−06 5.70E−05 8.00E−06 2.10E−054.30E−05 4.30E−05 −4.50E−05 −1.30E−05 2.40E−05 1.55 1.80E−05 1.00E−054.50E−05 1.50E−05 1.80E−05 4.60E−05 5.20E−05 −2.40E−05 −3.50E−053.70E−05 1.57 6.00E−06 1.00E−06 5.10E−05 1.70E−05 1.10E−05 5.20E−054.40E−05 −2.40E−05 −1.00E−05 1.10E−05 1.58 4.30E−05 2.00E−06 6.60E−051.80E−05 1.80E−05 4.40E−05 3.50E−05 −4.10E−05 −1.70E−05 3.20E−05 1.603.70E−05 1.00E−06 5.20E−05 5.00E−06 2.30E−05 5.00E−05 4.60E−05 −2.80E−05−1.70E−05 2.80E−05 1.62 1.70E−05 7.00E−06 5.20E−05 6.00E−06 1.50E−055.10E−05 4.10E−05 −3.70E−05 −3.00E−06 1.40E−05 1.63 3.50E−05 1.40E−054.50E−05 7.00E−06 4.10E−05 5.30E−05 3.40E−05 −3.80E−05 −1.00E−053.50E−05 1.65 2.20E−05 4.00E−06 4.40E−05 1.40E−05 2.60E−05 5.10E−054.70E−05 −5.00E−05 −2.10E−05 1.30E−05 1.67 1.20E−05 2.10E−05 5.50E−052.40E−05 2.00E−05 4.60E−05 4.10E−05 −3.10E−05 −2.10E−05 2.10E−05 1.682.30E−05 1.40E−05 5.20E−05 4.00E−06 4.00E−05 7.60E−05 3.70E−05 −2.80E−05−6.00E−06 3.10E−05 1.70 3.00E−06 1.80E−05 4.50E−05 1.70E−05 1.60E−055.30E−05 3.80E−05 −2.50E−05 −1.80E−05 1.50E−05 1.72 1.70E−05 1.70E−055.00E−05 2.10E−05 1.60E−05 6.10E−05 4.00E−05 −3.40E−05 −1.40E−051.30E−05 1.73 3.50E−05 8.00E−06 5.60E−05 1.30E−05 3.00E−05 5.00E−054.70E−05 −3.60E−05 −2.20E−05 7.00E−06 1.75 2.60E−05 7.00E−06 5.00E−052.00E−05 1.30E−05 5.70E−05 4.10E−05 −3.20E−05 −1.00E−05 2.50E−05 1.771.70E−05 1.10E−05 5.00E−05 2.40E−05 3.00E−05 5.60E−05 2.10E−05 −3.40E−05−2.00E−05 7.00E−06 1.78 3.10E−05 8.00E−06 4.60E−05 2.40E−05 3.40E−054.20E−05 3.50E−05 −3.00E−05 −3.10E−05 7.00E−06 1.80 4.00E−05 1.10E−054.50E−05 3.70E−05 4.00E−05 4.30E−05 3.20E−05 −3.40E−05 −3.10E−052.70E−05 1.82 1.80E−05 1.10E−05 4.00E−05 1.70E−05 2.60E−05 6.00E−054.00E−05 −2.20E−05 −2.70E−05 3.30E−05 1.83 7.00E−06 1.30E−05 5.30E−052.00E−05 1.30E−05 4.80E−05 4.60E−05 −4.10E−05 −5.00E−06 1.80E−05 1.852.10E−05 1.40E−05 6.10E−05 1.70E−05 1.70E−05 4.10E−05 5.50E−05 −4.00E−05−6.00E−06 1.70E−05 1.87 1.80E−05 1.80E−05 4.60E−05 2.70E−05 3.50E−053.60E−05 4.00E−05 −2.60E−05 −1.20E−05 2.10E−05 1.88 4.30E−05 2.50E−054.30E−05 1.80E−05 2.70E−05 5.00E−05 2.70E−05 −5.30E−05 −2.30E−053.10E−05 1.90 5.00E−05 2.20E−05 4.50E−05 2.40E−05 3.20E−05 5.50E−054.00E−05 −3.70E−05 −1.80E−05 1.30E−05 1.92 2.20E−05 1.70E−05 6.10E−052.40E−05 2.50E−05 4.60E−05 2.50E−05 −2.70E−05 −1.80E−05 2.60E−05 1.933.10E−05 2.50E−05 6.70E−05 2.80E−05 2.60E−05 4.10E−05 2.80E−05 −4.00E−05−2.50E−05 3.00E−05 1.95 2.70E−05 1.70E−05 6.30E−05 3.60E−05 2.50E−052.40E−05 3.00E−05 −4.70E−05 −2.80E−05 1.80E−05 1.97 2.40E−05 1.60E−054.60E−05 2.50E−05 2.70E−05 3.70E−05 1.80E−05 −4.20E−05 −3.00E−051.80E−05 1.98 3.20E−05 1.80E−05 3.30E−05 1.20E−05 1.80E−05 4.50E−054.00E−05 −4.60E−05 −1.10E−05 5.00E−05 2.00 3.10E−05 2.30E−05 5.10E−051.80E−05 2.30E−05 4.10E−05 3.60E−05 −4.00E−05 −1.20E−05 2.40E−05 2.022.70E−05 2.30E−05 4.10E−05 2.20E−05 1.70E−05 5.10E−05 2.40E−05 −3.30E−05−1.60E−05 3.60E−05 2.03 2.30E−05 2.00E−05 5.00E−05 2.00E−05 2.30E−055.10E−05 2.60E−05 −4.30E−05 −6.00E−06 3.20E−05 2.05 1.20E−05 2.20E−053.00E−05 2.80E−05 2.30E−05 4.70E−05 3.60E−05 −3.80E−05 −1.70E−053.60E−05 2.07 2.70E−05 1.70E−05 5.00E−05 1.40E−05 2.60E−05 4.30E−053.80E−05 −3.10E−05 −2.70E−05 3.70E−05 2.08 2.20E−05 2.70E−05 5.00E−051.30E−05 2.30E−05 5.50E−05 2.10E−05 −2.30E−05 −1.20E−05 3.30E−05 2.103.50E−05 2.30E−05 5.20E−05 1.70E−05 3.30E−05 3.40E−05 3.40E−05 −4.00E−05−2.50E−05 4.80E−05 2.12 2.20E−05 2.80E−05 3.80E−05 2.10E−05 2.40E−054.20E−05 2.50E−05 −3.40E−05 −2.50E−05 3.80E−05 2.13 1.70E−05 1.80E−055.30E−05 1.80E−05 2.40E−05 5.70E−05 2.10E−05 −6.00E−05 −2.30E−053.50E−05 2.15 2.20E−05 1.20E−05 5.40E−05 1.40E−05 2.80E−05 4.80E−052.50E−05 −4.30E−05 −1.60E−05 6.70E−05 2.17 3.40E−05 2.00E−05 4.60E−056.00E−06 2.80E−05 2.70E−05 2.00E−05 −4.00E−05 −1.10E−05 4.40E−05 2.183.20E−05 2.70E−05 6.10E−05 1.60E−05 2.70E−05 4.80E−05 3.20E−05 −4.80E−052.00E−06 4.50E−05 2.20 2.30E−05 2.40E−05 5.30E−05 1.80E−05 2.80E−055.00E−05 1.80E−05 −4.50E−05 −1.40E−05 3.10E−05 2.22 1.20E−05 2.10E−053.80E−05 7.00E−06 2.60E−05 5.20E−05 2.70E−05 −4.00E−05 −1.80E−053.20E−05 2.23 1.80E−05 7.00E−06 3.30E−05 −1.00E−06 4.70E−05 4.40E−052.10E−05 −4.00E−05 −2.80E−05 3.60E−05 2.25 2.60E−05 2.80E−05 4.20E−05−3.00E−06 3.20E−05 4.80E−05 2.40E−05 −4.20E−05 −1.70E−05 4.60E−05 2.272.50E−05 1.40E−05 5.00E−05 2.00E−05 3.20E−05 3.80E−05 5.00E−06 −2.60E−05−1.50E−05 4.10E−05 2.28 2.40E−05 1.30E−05 4.30E−05 8.00E−06 8.00E−065.70E−05 2.00E−05 −4.20E−05 −6.00E−06 3.10E−05 2.30 3.20E−05 3.00E−054.60E−05 −1.00E−06 3.40E−05 4.40E−05 2.30E−05 −3.80E−05 −2.60E−051.60E−05 2.32 2.60E−05 2.70E−05 3.00E−05 1.10E−05 4.10E−05 5.30E−051.60E−05 −1.80E−05 −1.60E−05 2.10E−05 2.33 1.60E−05 2.00E−05 4.00E−052.00E−05 3.40E−05 4.60E−05 2.20E−05 −3.70E−05 −1.30E−05 2.20E−05 2.351.80E−05 2.80E−05 3.80E−05 5.00E−06 3.60E−05 5.00E−05 2.40E−05 −2.60E−05−2.10E−05 2.30E−05 2.37 2.50E−05 3.30E−05 3.80E−05 1.70E−05 3.70E−055.00E−05 2.50E−05 −4.20E−05 −8.00E−06 1.70E−05 2.38 3.20E−05 2.80E−054.00E−05 1.30E−05 4.60E−05 6.20E−05 2.50E−05 −5.60E−05 −2.50E−052.30E−05 2.40 2.40E−05 2.20E−05 4.50E−05 7.00E−06 4.00E−05 5.50E−053.50E−05 −3.30E−05 −2.00E−05 3.00E−05 2.42 1.60E−05 2.40E−05 4.80E−05−1.60E−05 3.10E−05 5.50E−05 2.50E−05 −3.70E−05 −6.00E−06 2.40E−05 2.434.00E−05 2.20E−05 3.30E−05 1.00E−05 3.00E−05 5.80E−05 1.70E−05 −3.20E−05−1.60E−05 2.00E−05 2.45 3.00E−05 1.30E−05 4.70E−05 1.50E−05 2.40E−055.00E−05 3.70E−05 −3.20E−05 −7.00E−06 3.70E−05 2.47 2.80E−05 2.50E−053.80E−05 4.00E−06 5.20E−05 6.20E−05 3.40E−05 −3.40E−05 0.00E+00 2.20E−052.48 1.70E−05 1.80E−05 4.50E−05 2.00E−06 3.80E−05 6.60E−05 1.80E−05−4.70E−05 −1.30E−05 2.00E−05 2.50 1.80E−05 7.00E−06 3.30E−05 1.70E−053.80E−05 5.20E−05 2.50E−05 −4.80E−05 −8.00E−06 2.40E−05 2.52 2.60E−052.60E−05 5.10E−05 2.00E−06 2.50E−05 6.40E−05 2.30E−05 −4.60E−05−7.00E−06 2.50E−05 2.53 4.50E−05 2.20E−05 3.60E−05 4.00E−06 3.10E−054.10E−05 3.00E−05 −3.70E−05 −2.20E−05 1.50E−05 2.55 2.10E−05 1.60E−053.80E−05 4.00E−06 5.30E−05 4.80E−05 1.70E−05 −3.40E−05 −5.00E−062.20E−05 2.57 3.00E−05 1.80E−05 3.30E−05 −6.00E−06 4.50E−05 4.20E−052.60E−05 −4.50E−05 8.00E−06 1.30E−05 2.58 2.80E−05 2.70E−05 3.60E−05−1.00E−06 4.30E−05 4.40E−05 2.20E−05 −3.10E−05 1.30E−05 2.00E−05 2.603.40E−05 1.80E−05 4.70E−05 3.00E−06 4.70E−05 5.30E−05 2.70E−05 −3.30E−051.00E−05 2.30E−05 2.62 3.60E−05 2.60E−05 4.40E−05 −1.00E−05 4.80E−055.10E−05 2.50E−05 −4.10E−05 1.60E−05 2.20E−05 2.63 2.30E−05 2.10E−053.30E−05 1.00E−06 4.50E−05 3.70E−05 1.20E−05 −3.40E−05 −6.00E−062.30E−05 2.65 2.00E−05 3.00E−05 2.40E−05 −1.00E−06 4.50E−05 4.30E−051.80E−05 −5.20E−05 2.00E−06 1.70E−05 2.67 1.40E−05 3.00E−05 3.30E−058.00E−06 3.40E−05 5.80E−05 2.00E−06 −5.70E−05 1.20E−05 1.00E−05 2.684.00E−05 1.40E−05 4.40E−05 −1.40E−05 3.00E−05 4.30E−05 1.50E−05−3.50E−05 4.80E−05 2.30E−05 2.70 2.20E−05 2.60E−05 3.70E−05 −6.00E−065.70E−05 4.60E−05 8.00E−06 −4.50E−05 4.00E−06 1.80E−05 2.72 2.50E−051.70E−05 3.40E−05 1.00E−06 4.50E−05 3.70E−05 5.00E−06 −4.30E−05 1.40E−051.50E−05 2.73 3.10E−05 2.40E−05 5.40E−05 −1.40E−05 3.10E−05 4.50E−052.00E−05 −5.00E−05 −4.00E−06 2.50E−05 2.75 1.10E−05 1.80E−05 3.10E−05−2.00E−06 4.00E−05 4.20E−05 1.20E−05 −2.80E−05 1.50E−05 1.10E−05 2.771.70E−05 2.70E−05 4.80E−05 −1.10E−05 3.60E−05 2.80E−05 1.60E−05−3.40E−05 1.10E−05 2.00E−06 2.78 2.00E−05 4.40E−05 4.80E−05 1.10E−054.60E−05 4.70E−05 1.10E−05 −4.70E−05 −3.00E−06 1.80E−05 2.80 3.80E−052.00E−05 4.60E−05 −7.00E−06 3.80E−05 3.40E−05 1.50E−05 −3.60E−051.80E−05 8.00E−06 2.82 3.50E−05 3.70E−05 3.20E−05 −5.00E−06 4.00E−053.60E−05 1.50E−05 −3.40E−05 1.40E−05 1.70E−05 2.83 4.80E−05 1.60E−054.00E−05 −8.00E−06 3.40E−05 3.40E−05 1.30E−05 −3.20E−05 1.10E−058.00E−06 2.85 1.50E−05 1.30E−05 4.10E−05 −7.00E−06 5.70E−05 2.40E−051.60E−05 −3.70E−05 4.00E−06 2.00E−05 2.87 1.50E−05 4.70E−05 3.70E−05−1.00E−05 3.60E−05 3.50E−05 8.00E−06 −4.10E−05 1.00E−05 2.00E−05 2.883.00E−05 2.70E−05 3.60E−05 −1.80E−05 3.10E−05 4.00E−05 1.10E−05−4.80E−05 −3.00E−06 2.20E−05 2.90 2.60E−05 2.00E−05 3.80E−05 −6.00E−065.30E−05 7.70E−05 4.00E−06 −3.00E−05 −1.40E−05 3.20E−05 2.92 1.10E−052.40E−05 3.60E−05 −1.60E−05 4.60E−05 2.70E−05 5.00E−06 −4.50E−053.00E−06 1.30E−05 2.93 2.70E−05 8.00E−06 3.20E−05 0.00E+00 5.50E−053.20E−05 5.00E−06 −3.30E−05 3.00E−06 3.00E−05 2.95 3.50E−05 1.40E−053.70E−05 −1.20E−05 4.10E−05 3.80E−05 1.10E−05 −4.40E−05 2.00E−061.40E−05 2.97 5.10E−05 1.40E−05 4.10E−05 −1.20E−05 2.80E−05 5.70E−055.00E−06 −4.10E−05 −1.10E−05 1.70E−05 2.98 1.70E−05 1.70E−05 3.50E−05−1.10E−05 5.00E−05 5.60E−05 7.00E−06 −3.80E−05 4.00E−06 7.00E−06 3.002.50E−05 1.50E−05 3.60E−05 −1.50E−05 4.50E−05 3.20E−05 1.00E−05−4.50E−05 −1.80E−05 8.00E−06 3.02 3.30E−05 1.60E−05 4.60E−05 4.00E−064.10E−05 4.20E−05 −1.20E−05 −4.50E−05 −1.00E−06 2.20E−05 3.03 1.50E−051.30E−05 5.70E−05 −1.10E−05 5.00E−05 3.10E−05 1.00E−05 −5.00E−051.00E−06 3.60E−05 3.05 2.20E−05 1.40E−05 2.20E−05 −1.00E−05 5.20E−053.20E−05 −1.00E−06 −4.20E−05 −1.20E−05 1.00E−05 3.07 2.60E−05 2.40E−054.50E−05 −1.20E−05 4.20E−05 3.20E−05 1.00E−06 −2.70E−05 −1.10E−051.80E−05 3.08 1.00E−05 1.20E−05 2.60E−05 −1.00E−06 4.30E−05 3.80E−058.00E−06 −4.50E−05 −6.00E−06 2.10E−05 3.10 7.00E−06 1.70E−05 3.20E−05−5.00E−06 5.70E−05 4.00E−05 1.30E−05 −5.30E−05 −1.20E−05 2.70E−05 3.121.00E−05 1.60E−05 3.10E−05 −2.20E−05 4.20E−05 4.50E−05 −4.00E−06−4.60E−05 −3.00E−06 1.50E−05 3.13 2.40E−05 1.00E−05 3.80E−05 −1.50E−054.30E−05 3.20E−05 1.60E−05 −4.30E−05 0.00E+00 2.80E−05 3.15 2.60E−052.40E−05 3.40E−05 1.00E−06 4.80E−05 3.10E−05 5.00E−06 −2.50E−05−1.20E−05 2.20E−05 3.17 8.00E−06 3.20E−05 2.80E−05 0.00E+00 4.80E−055.00E−05 1.00E−06 −4.20E−05 −1.30E−05 7.00E−06 3.18 1.80E−05 1.80E−053.10E−05 −1.70E−05 4.60E−05 4.60E−05 0.00E+00 −4.10E−05 −8.00E−061.50E−05 3.20 1.30E−05 2.20E−05 3.10E−05 −7.00E−06 4.20E−05 4.00E−051.40E−05 −4.40E−05 −7.00E−06 2.00E−05 3.22 3.50E−05 2.60E−05 5.30E−053.00E−06 5.30E−05 3.40E−05 −1.00E−06 −3.70E−05 −1.40E−05 1.80E−05 3.232.40E−05 2.20E−05 4.50E−05 −1.10E−05 4.10E−05 3.70E−05 1.10E−05−4.00E−05 −1.00E−06 3.00E−05 3.25 1.80E−05 1.10E−05 3.60E−05 −5.00E−067.40E−05 3.80E−05 1.80E−05 −2.80E−05 −6.00E−06 2.80E−05 3.27 2.50E−051.50E−05 2.60E−05 −5.00E−06 4.80E−05 5.10E−05 6.00E−06 −4.30E−05−7.00E−06 1.40E−05 3.28 1.40E−05 0.00E+00 5.00E−05 −1.00E−05 4.10E−054.30E−05 7.00E−06 −3.80E−05 −2.00E−06 1.80E−05 3.30 3.10E−05 1.10E−052.70E−05 −1.40E−05 3.10E−05 5.20E−05 1.20E−05 −4.40E−05 −8.00E−063.20E−05 3.32 2.30E−05 7.00E−06 3.20E−05 −1.00E−06 3.50E−05 3.70E−051.20E−05 −5.10E−05 −1.00E−06 3.60E−05 3.33 2.60E−05 4.00E−06 2.70E−05−2.00E−06 3.60E−05 5.00E−05 −2.00E−06 −5.20E−05 −4.00E−06 3.10E−05 3.352.60E−05 1.80E−05 3.50E−05 −1.00E−05 4.30E−05 5.50E−05 2.00E−06−5.80E−05 7.00E−06 2.10E−05 3.37 8.00E−06 2.70E−05 5.00E−05 −1.50E−053.60E−05 4.20E−05 1.10E−05 −5.50E−05 1.80E−05 1.80E−05 3.38 2.80E−051.80E−05 3.40E−05 −1.80E−05 2.80E−05 3.20E−05 −1.30E−05 −4.00E−05−1.50E−05 2.80E−05 3.40 2.40E−05 1.30E−05 3.40E−05 0.00E+00 2.50E−053.50E−05 1.80E−05 −3.70E−05 −3.00E−06 2.00E−05 3.42 1.70E−05 3.10E−054.10E−05 −4.00E−06 3.60E−05 4.10E−05 2.00E−06 −4.00E−05 0.00E+003.00E−05 3.43 1.60E−05 5.00E−06 3.70E−05 −1.00E−06 5.10E−05 5.60E−051.20E−05 −1.80E−05 8.00E−06 2.70E−05 3.45 2.60E−05 2.00E−05 4.00E−05−3.00E−06 3.20E−05 3.50E−05 7.00E−06 −4.30E−05 8.00E−06 2.40E−05 3.472.30E−05 7.00E−06 3.20E−05 1.00E−06 3.20E−05 2.60E−05 −2.00E−06−5.00E−05 0.00E+00 4.60E−05 3.48 1.80E−05 3.00E−06 3.50E−05 6.00E−065.80E−05 3.60E−05 5.00E−06 −5.50E−05 7.00E−06 3.20E−05 3.50 2.20E−051.10E−05 3.50E−05 −4.00E−06 4.60E−05 4.40E−05 1.10E−05 −5.70E−051.00E−05 2.50E−05 3.52 2.00E−05 2.10E−05 3.60E−05 1.50E−05 5.10E−053.40E−05 5.00E−06 −3.20E−05 7.00E−06 3.30E−05 3.53 1.50E−05 3.20E−054.30E−05 1.00E−05 2.80E−05 5.10E−05 1.00E−06 −4.80E−05 −8.00E−063.30E−05 3.55 2.00E−05 1.80E−05 3.10E−05 1.80E−05 3.70E−05 3.30E−05−5.00E−06 −5.80E−05 1.00E−06 3.30E−05 3.57 2.20E−05 1.20E−05 4.60E−058.00E−06 2.50E−05 4.50E−05 1.50E−05 −3.50E−05 1.00E−05 1.80E−05 3.581.50E−05 8.00E−06 3.30E−05 2.70E−05 3.30E−05 4.40E−05 8.00E−06 −4.40E−054.00E−06 3.60E−05 3.60 2.10E−05 6.00E−06 4.50E−05 2.60E−05 4.50E−053.40E−05 6.00E−06 −4.20E−05 3.00E−06 3.60E−05 3.62 1.20E−05 1.30E−054.40E−05 3.00E−05 4.40E−05 4.20E−05 6.00E−06 −4.30E−05 −1.30E−052.10E−05 3.63 1.70E−05 2.50E−05 3.60E−05 3.50E−05 3.40E−05 4.80E−05−1.00E−05 −4.30E−05 −3.00E−06 2.60E−05 3.65 1.80E−05 2.20E−05 4.20E−056.50E−05 3.00E−05 4.30E−05 1.50E−05 −4.80E−05 2.00E−06 3.30E−05 3.672.20E−05 1.80E−05 3.60E−05 4.70E−05 3.70E−05 3.00E−05 1.10E−05 −4.30E−050.00E+00 3.20E−05 3.68 6.00E−06 2.00E−06 2.60E−05 4.60E−05 3.30E−053.30E−05 3.00E−06 −4.40E−05 1.30E−05 4.20E−05 3.70 2.00E−06 2.20E−053.10E−05 6.10E−05 5.00E−05 3.30E−05 1.00E−05 −4.10E−05 −5.00E−061.30E−05 3.72 1.80E−05 1.60E−05 2.80E−05 6.40E−05 3.00E−05 4.70E−058.00E−06 −5.10E−05 2.00E−06 3.60E−05 3.73 2.30E−05 2.40E−05 2.70E−056.90E−05 3.30E−05 4.80E−05 5.00E−06 −4.40E−05 8.00E−06 2.50E−05 3.752.20E−05 1.30E−05 4.10E−05 7.20E−05 3.10E−05 4.10E−05 1.00E−05 0.00E+003.40E−05 3.40E−05 3.77 2.40E−05 2.00E−06 4.10E−05 7.00E−05 4.30E−054.50E−05 1.00E−05 −5.20E−05 4.00E−06 4.10E−05 3.78 −1.00E−06 2.30E−053.10E−05 1.10E−04 2.40E−05 4.40E−05 1.70E−05 −5.80E−05 3.20E−05 4.50E−053.80 2.60E−05 1.10E−05 3.40E−05 7.20E−05 4.00E−05 4.10E−05 1.30E−05−4.80E−05 1.00E−05 4.30E−05 3.82 2.40E−05 2.80E−05 3.00E−05 6.20E−053.40E−05 4.80E−05 1.00E−05 −6.10E−05 2.00E−06 2.40E−05 3.83 1.40E−051.80E−05 3.30E−05 5.20E−05 2.30E−05 4.00E−05 1.80E−05 −2.80E−05 6.00E−063.60E−05 3.85 2.00E−05 8.00E−06 3.20E−05 6.20E−05 3.40E−05 5.00E−051.40E−05 −4.30E−05 2.00E−05 4.30E−05 3.87 1.30E−05 5.00E−06 3.80E−054.40E−05 1.70E−05 4.60E−05 5.00E−06 −5.00E−05 2.20E−05 4.00E−05 3.881.40E−05 7.00E−06 4.20E−05 4.50E−05 2.60E−05 4.70E−05 2.10E−05 −5.50E−051.30E−05 2.80E−05 3.90 2.30E−05 1.40E−05 4.10E−05 3.70E−05 2.50E−056.00E−05 2.00E−06 −4.80E−05 1.20E−05 4.00E−05 3.92 1.40E−05 1.60E−053.80E−05 4.60E−05 3.00E−05 6.70E−05 2.80E−05 −6.00E−05 1.20E−05 4.40E−053.93 1.40E−05 1.40E−05 3.80E−05 3.40E−05 1.70E−05 7.40E−05 1.70E−05−4.30E−05 2.30E−05 3.20E−05 3.95 1.80E−05 2.60E−05 3.50E−05 3.70E−053.40E−05 7.20E−05 3.10E−05 −5.60E−05 1.80E−05 3.10E−05 3.97 2.30E−052.30E−05 4.20E−05 3.30E−05 3.80E−05 8.00E−05 2.80E−05 −5.70E−05 1.20E−054.40E−05 3.98 1.30E−05 1.70E−05 3.70E−05 4.20E−05 3.30E−05 9.00E−053.30E−05 −6.30E−05 1.70E−05 3.50E−05 4.00 4.10E−05 1.20E−05 4.30E−052.00E−05 2.30E−05 1.01E−04 4.50E−05 −4.70E−05 3.50E−05 3.60E−05 4.028.00E−06 6.00E−06 3.30E−05 3.00E−05 4.00E−05 1.06E−04 5.40E−05 −5.20E−051.10E−05 3.50E−05 4.03 2.20E−05 8.00E−06 2.40E−05 1.00E−05 2.30E−051.09E−04 6.20E−05 −6.50E−05 2.20E−05 5.00E−05 4.05 1.70E−05 3.00E−064.00E−05 2.10E−05 3.30E−05 1.07E−04 7.90E−05 −3.00E−05 1.00E−05 3.20E−054.07 2.40E−05 1.00E−05 4.10E−05 1.70E−05 3.10E−05 1.11E−04 8.00E−05−5.30E−05 1.50E−05 2.40E−05 4.08 2.40E−05 1.00E−05 3.60E−05 1.20E−051.80E−05 1.19E−04 9.00E−05 −5.30E−05 2.20E−05 3.70E−05 4.10 2.50E−052.30E−05 1.70E−05 2.10E−05 1.60E−05 1.11E−04 1.16E−04 −3.40E−05 1.70E−053.80E−05 4.12 1.70E−05 2.30E−05 4.40E−05 8.00E−06 3.30E−05 1.21E−049.90E−05 −5.40E−05 1.00E−05 4.00E−05 4.13 1.80E−05 1.40E−05 2.80E−052.50E−05 2.20E−05 1.05E−04 1.06E−04 −6.70E−05 1.80E−05 4.00E−05 4.152.30E−05 1.40E−05 3.80E−05 2.40E−05 2.10E−05 1.17E−04 1.07E−04 −3.60E−052.00E−06 4.80E−05 4.17 5.00E−06 7.00E−06 3.10E−05 1.30E−05 1.80E−051.11E−04 1.19E−04 −4.20E−05 1.10E−05 5.20E−05 4.18 2.50E−05 2.40E−053.20E−05 −5.00E−06 1.40E−05 1.10E−04 1.16E−04 −4.50E−05 8.00E−064.50E−05 4.20 2.30E−05 4.00E−06 2.10E−05 1.10E−05 3.20E−05 1.16E−041.26E−04 −5.00E−05 8.00E−06 5.00E−05 4.22 2.60E−05 8.00E−06 4.70E−052.00E−06 2.80E−05 9.40E−05 1.36E−04 −5.40E−05 2.10E−05 4.80E−05 4.232.30E−05 4.00E−06 2.70E−05 5.00E−06 3.00E−05 1.01E−04 1.33E−04 −5.50E−051.30E−05 4.60E−05 4.25 2.80E−05 7.00E−06 3.60E−05 −6.00E−06 2.50E−059.40E−05 1.32E−04 −5.80E−05 2.40E−05 5.20E−05 4.27 1.00E−05 5.00E−063.30E−05 7.00E−06 2.20E−05 7.40E−05 1.26E−04 −5.00E−05 1.50E−05 4.10E−054.28 1.60E−05 5.00E−06 3.00E−05 2.00E−06 2.10E−05 8.50E−05 1.39E−04−6.50E−05 2.20E−05 3.70E−05 4.30 2.10E−05 3.00E−06 3.70E−05 2.00E−061.80E−05 6.70E−05 9.70E−05 −5.20E−05 1.60E−05 2.50E−05 4.32 2.30E−051.10E−05 3.70E−05 −8.00E−06 2.60E−05 8.20E−05 9.70E−05 −4.70E−051.80E−05 5.80E−05 4.33 3.30E−05 −2.00E−06 4.70E−05 −3.00E−06 2.60E−056.60E−05 8.60E−05 −5.50E−05 1.10E−05 4.20E−05 4.35 2.40E−05 7.00E−065.00E−05 −7.00E−06 2.20E−05 6.10E−05 9.40E−05 −6.30E−05 4.00E−064.60E−05 4.37 1.50E−05 1.40E−05 3.10E−05 0.00E+00 3.20E−05 5.60E−058.20E−05 −5.70E−05 1.30E−05 4.40E−05 4.38 2.80E−05 5.00E−06 4.70E−051.40E−05 1.10E−05 5.40E−05 7.70E−05 −5.60E−05 8.00E−06 2.70E−05 4.401.60E−05 1.00E−05 5.20E−05 −5.00E−06 3.70E−05 4.70E−05 7.50E−05−4.70E−05 1.10E−05 5.00E−05 4.42 2.70E−05 1.20E−05 4.40E−05 −6.00E−061.70E−05 6.00E−05 7.40E−05 −4.80E−05 1.60E−05 4.10E−05 4.43 3.10E−052.40E−05 4.60E−05 0.00E+00 1.70E−05 3.60E−05 6.20E−05 −6.20E−05 7.00E−063.50E−05 4.45 2.40E−05 1.30E−05 4.70E−05 7.00E−06 1.00E−05 3.70E−056.20E−05 −6.10E−05 −1.00E−06 3.10E−05 4.47 4.40E−05 5.00E−06 4.80E−05−3.00E−06 1.30E−05 5.10E−05 5.50E−05 −4.10E−05 3.70E−05 3.40E−05 4.482.10E−05 −1.00E−06 4.50E−05 −1.20E−05 1.60E−05 4.50E−05 5.60E−05−5.10E−05 3.00E−06 4.50E−05 4.50 2.50E−05 1.20E−05 4.30E−05 −7.00E−061.20E−05 4.50E−05 6.00E−05 −5.00E−05 1.30E−05 5.40E−05 4.52 2.40E−052.30E−05 5.60E−05 −1.00E−05 1.70E−05 3.20E−05 5.30E−05 −5.80E−052.20E−05 5.00E−05 4.53 3.60E−05 6.00E−06 5.80E−05 −1.00E−05 1.20E−054.60E−05 5.10E−05 −5.80E−05 8.00E−06 5.10E−05 4.55 3.20E−05 2.50E−054.10E−05 3.00E−06 1.60E−05 2.80E−05 4.50E−05 −5.50E−05 2.00E−05 4.80E−054.57 2.70E−05 4.00E−06 5.50E−05 −1.00E−05 4.00E−06 1.70E−05 5.10E−05−5.00E−05 2.00E−06 6.00E−05 4.58 3.80E−05 0.00E+00 4.80E−05 1.00E−061.50E−05 7.20E−05 4.10E−05 −3.80E−05 1.00E−05 3.70E−05 4.60 1.80E−05−3.00E−06 5.50E−05 −5.00E−06 6.00E−06 3.10E−05 5.40E−05 −5.30E−054.00E−06 4.10E−05 4.62 1.40E−05 3.00E−06 5.10E−05 −1.00E−06 5.00E−061.80E−05 4.40E−05 −6.10E−05 1.30E−05 4.70E−05 4.63 1.20E−05 1.30E−055.10E−05 −1.10E−05 6.00E−06 3.00E−05 4.30E−05 −6.00E−05 1.00E−065.60E−05 4.65 2.40E−05 2.40E−05 4.30E−05 −7.00E−06 2.10E−05 2.40E−052.80E−05 −6.70E−05 1.10E−05 5.30E−05 4.67 2.20E−05 2.30E−05 5.40E−05−1.60E−05 3.10E−05 2.20E−05 4.70E−05 −6.00E−05 1.00E−06 4.20E−05 4.681.60E−05 8.00E−06 5.40E−05 −1.20E−05 1.50E−05 2.20E−05 4.60E−05−6.90E−05 1.50E−05 3.80E−05 4.70 2.60E−05 1.70E−05 4.30E−05 −2.00E−065.00E−06 5.00E−06 4.00E−05 −5.50E−05 5.00E−06 4.70E−05 4.72 2.00E−05−5.00E−06 5.80E−05 4.00E−06 −1.00E−06 2.10E−05 2.40E−05 −5.30E−051.10E−05 4.70E−05 4.73 2.50E−05 1.80E−05 6.10E−05 −1.60E−05 1.30E−052.00E−05 4.00E−05 −4.10E−05 1.60E−05 4.10E−05 4.75 2.00E−05 −2.00E−065.70E−05 −1.10E−05 4.00E−06 8.00E−06 2.30E−05 −5.00E−05 1.10E−053.70E−05 4.77 2.30E−05 0.00E+00 5.70E−05 2.00E−06 1.80E−05 −3.00E−063.30E−05 −5.40E−05 7.00E−06 6.00E−05 4.78 1.50E−05 1.50E−05 5.20E−05−2.00E−06 1.20E−05 7.00E−06 2.80E−05 −5.40E−05 4.00E−06 4.60E−05 4.802.10E−05 2.10E−05 4.70E−05 0.00E+00 1.30E−05 2.20E−05 3.10E−05 −3.50E−051.20E−05 4.10E−05 4.82 2.50E−05 3.00E−05 6.40E−05 1.00E−06 7.00E−061.60E−05 1.50E−05 −5.00E−05 1.60E−05 4.10E−05 4.83 7.00E−06 2.00E−056.50E−05 −5.00E−06 2.30E−05 1.00E−05 2.60E−05 −5.60E−05 2.00E−054.30E−05 4.85 2.10E−05 1.00E−05 5.60E−05 −3.00E−06 1.10E−05 8.00E−063.50E−05 −6.10E−05 5.00E−06 4.40E−05 4.87 2.70E−05 8.00E−06 5.50E−053.00E−06 −4.00E−06 1.10E−05 2.40E−05 −5.70E−05 8.00E−06 4.80E−05 4.881.70E−05 2.00E−06 5.40E−05 −3.00E−06 0.00E+00 1.40E−05 2.00E−05−5.00E−05 −2.00E−06 5.10E−05 4.90 1.20E−05 7.00E−06 5.10E−05 3.00E−061.40E−05 −4.00E−06 1.80E−05 −6.20E−05 1.60E−05 3.80E−05 4.92 3.50E−051.30E−05 5.20E−05 1.00E−06 1.10E−05 1.10E−05 3.70E−05 −5.30E−05 6.00E−065.60E−05 4.93 3.20E−05 1.60E−05 6.00E−05 −1.20E−05 3.00E−06 −2.00E−061.30E−05 −6.30E−05 −1.00E−06 5.10E−05 4.95 2.30E−05 8.00E−06 6.30E−050.00E+00 1.50E−05 −1.00E−06 1.30E−05 −2.60E−05 1.20E−05 4.10E−05 4.971.50E−05 7.00E−06 7.00E−05 1.00E−05 8.00E−06 8.00E−06 2.20E−05 −5.60E−051.00E−05 4.80E−05 4.98 1.70E−05 3.00E−06 4.60E−05 1.00E−05 1.80E−05−1.00E−06 1.80E−05 −7.30E−05 3.00E−06 5.70E−05 5.00 2.20E−05 1.70E−055.20E−05 −1.00E−06 0.00E+00 1.20E−05 2.20E−05 −5.70E−05 1.40E−055.50E−05 5.02 1.80E−05 1.20E−05 6.00E−05 1.30E−05 7.00E−06 3.00E−062.20E−05 −5.30E−05 −2.00E−06 5.20E−05 5.03 2.00E−05 1.50E−05 5.10E−052.00E−06 1.30E−05 2.00E−06 1.70E−05 −4.60E−05 7.00E−06 4.70E−05 5.051.70E−05 1.00E−05 5.40E−05 1.00E−05 1.30E−05 5.00E−06 2.60E−05 −5.80E−051.10E−05 6.30E−05 5.07 3.30E−05 2.00E−06 6.10E−05 −2.00E−06 3.00E−067.00E−06 1.60E−05 −5.30E−05 1.80E−05 6.00E−05 5.08 2.70E−05 2.10E−055.00E−05 1.20E−05 1.00E−06 −1.60E−05 1.50E−05 −6.20E−05 −3.00E−065.50E−05 5.10 1.70E−05 2.20E−05 4.50E−05 3.00E−06 1.20E−05 −1.00E−064.00E−05 −6.00E−05 5.00E−06 6.50E−05 5.12 1.50E−05 6.00E−06 6.40E−050.00E+00 1.00E−05 −1.40E−05 1.70E−05 −4.80E−05 −2.00E−06 5.70E−05 5.132.30E−05 7.00E−06 5.80E−05 1.10E−05 7.00E−06 4.00E−06 1.60E−05 −8.50E−058.00E−06 4.80E−05 5.15 2.10E−05 1.10E−05 7.00E−05 4.00E−06 1.80E−05−1.00E−06 1.50E−05 −5.40E−05 6.00E−06 5.40E−05 5.17 2.00E−05 1.40E−055.30E−05 2.60E−05 1.60E−05 0.00E+00 2.40E−05 −7.00E−05 −1.40E−055.50E−05 5.18 1.60E−05 6.00E−06 7.10E−05 5.00E−06 5.00E−06 0.00E+001.70E−05 −6.50E−05 3.00E−06 5.80E−05 5.20 3.00E−06 6.00E−06 6.40E−055.00E−06 2.00E−06 4.00E−06 1.40E−05 −6.10E−05 1.40E−05 5.70E−05 5.222.50E−05 2.80E−05 5.50E−05 2.40E−05 1.30E−05 3.00E−06 1.40E−05 −5.30E−050.00E+00 4.60E−05 5.23 1.20E−05 2.70E−05 6.70E−05 8.00E−06 1.30E−051.30E−05 1.60E−05 −5.10E−05 1.00E−05 6.20E−05 5.25 2.80E−05 1.80E−056.00E−05 3.00E−06 −3.00E−06 −5.00E−06 1.80E−05 −6.90E−05 1.10E−055.80E−05 5.27 1.20E−05 1.10E−05 6.50E−05 2.60E−05 5.00E−06 5.00E−062.80E−05 −6.50E−05 1.30E−05 5.30E−05 5.28 1.30E−05 4.00E−06 7.60E−051.10E−05 1.40E−05 1.10E−05 1.60E−05 −5.00E−05 4.00E−06 6.10E−05 5.302.10E−05 8.00E−06 5.20E−05 1.20E−05 −1.00E−06 −5.00E−06 1.20E−05−5.80E−05 8.00E−06 4.20E−05 5.32 2.60E−05 1.00E−05 6.30E−05 2.80E−051.50E−05 4.00E−06 2.30E−05 −5.30E−05 0.00E+00 5.40E−05 5.33 1.80E−052.10E−05 6.50E−05 2.00E−05 1.10E−05 −3.00E−06 1.40E−05 −5.30E−051.10E−05 4.50E−05 5.35 1.70E−05 1.20E−05 6.70E−05 1.20E−05 −8.00E−067.00E−06 1.40E−05 −5.60E−05 −8.00E−06 6.50E−05 5.37 1.80E−05 3.00E−066.40E−05 3.10E−05 7.00E−06 −4.00E−06 2.10E−05 −7.70E−05 −2.00E−064.40E−05 5.38 1.60E−05 2.00E−05 6.50E−05 1.40E−05 2.40E−05 −2.00E−062.50E−05 −7.00E−05 −4.00E−06 6.60E−05 5.40 1.30E−05 1.20E−05 5.10E−055.00E−06 7.00E−06 −2.00E−06 1.50E−05 −6.60E−05 6.00E−06 4.50E−05 5.423.80E−05 2.40E−05 7.60E−05 1.10E−05 −1.00E−06 −7.00E−06 6.00E−06−5.80E−05 7.00E−06 5.10E−05 5.43 6.00E−06 7.00E−06 7.70E−05 2.00E−058.00E−06 2.00E−06 2.10E−05 −6.20E−05 −5.00E−06 4.50E−05 5.45 1.30E−051.20E−05 7.00E−05 1.30E−05 1.20E−05 −1.00E−06 2.60E−05 −4.70E−051.40E−05 6.00E−05 5.47 1.80E−05 1.30E−05 7.50E−05 8.00E−06 1.30E−052.00E−06 2.40E−05 −5.40E−05 7.00E−06 5.50E−05 5.48 1.20E−05 2.20E−056.30E−05 1.30E−05 7.00E−06 −7.00E−06 2.00E−05 −6.00E−05 8.00E−065.00E−05 5.50 1.60E−05 3.20E−05 7.40E−05 2.60E−05 8.00E−06 4.00E−062.00E−05 −5.20E−05 1.40E−05 6.30E−05 5.52 1.80E−05 2.40E−05 4.80E−058.00E−06 5.00E−06 −4.00E−06 1.80E−05 −5.80E−05 1.40E−05 4.70E−05 5.532.30E−05 2.00E−05 6.00E−05 3.00E−06 5.00E−06 7.00E−06 1.80E−05 −3.60E−051.00E−06 5.00E−05 5.55 2.40E−05 1.30E−05 7.50E−05 2.20E−05 7.00E−063.00E−06 2.20E−05 −5.40E−05 1.00E−06 6.00E−05 5.57 2.80E−05 1.60E−056.90E−05 1.50E−05 1.20E−05 1.80E−05 1.30E−05 −5.40E−05 4.00E−06 4.40E−055.58 3.00E−06 2.40E−05 7.10E−05 2.50E−05 2.00E−06 −8.00E−06 2.10E−05−6.50E−05 1.00E−05 6.50E−05 5.60 2.00E−05 2.30E−05 5.40E−05 2.10E−05−1.00E−05 5.00E−06 1.70E−05 −5.40E−05 0.00E+00 5.50E−05 5.62 1.70E−052.00E−05 6.90E−05 1.60E−05 5.00E−06 1.00E−06 2.20E−05 −5.10E−05 6.00E−065.80E−05 5.63 2.00E−05 1.30E−05 5.50E−05 3.00E−05 −6.00E−06 −1.10E−053.50E−05 −3.40E−05 −4.00E−06 5.30E−05 5.65 2.00E−05 1.40E−05 7.70E−051.10E−05 0.00E+00 −4.00E−06 1.30E−05 −4.20E−05 2.00E−06 4.50E−05 5.674.00E−06 3.10E−05 7.20E−05 2.50E−05 −1.00E−06 −4.00E−06 2.80E−05−5.20E−05 8.00E−06 4.60E−05 5.68 1.30E−05 2.10E−05 6.10E−05 8.00E−062.00E−06 0.00E+00 2.40E−05 −3.60E−05 5.00E−06 5.50E−05 5.70 1.10E−052.50E−05 7.30E−05 1.70E−05 6.00E−06 −6.00E−06 1.80E−05 −4.30E−058.00E−06 4.20E−05 5.72 3.50E−05 5.00E−06 6.40E−05 1.70E−05 5.00E−06−1.00E−06 2.00E−05 −4.10E−05 −2.00E−06 5.00E−05 5.73 1.30E−05 2.20E−056.20E−05 2.10E−05 8.00E−06 −1.00E−05 2.30E−05 −5.00E−05 4.00E−067.30E−05 5.75 2.20E−05 1.70E−05 7.20E−05 2.60E−05 1.00E−06 2.00E−051.80E−05 −5.10E−05 −1.20E−05 4.00E−05 5.77 6.00E−06 2.80E−05 6.20E−051.00E−05 1.00E−06 −2.00E−06 2.40E−05 −4.40E−05 −1.00E−06 4.10E−05 5.782.00E−05 2.50E−05 4.80E−05 2.20E−05 8.00E−06 2.00E−06 2.20E−05 −3.10E−050.00E+00 4.50E−05 5.80 2.00E−05 1.80E−05 6.50E−05 1.30E−05 8.00E−061.00E−06 1.10E−05 −5.10E−05 −8.00E−06 3.80E−05 5.82 1.10E−05 1.80E−055.50E−05 8.00E−06 4.00E−06 0.00E+00 1.40E−05 −4.10E−05 2.00E−06 4.00E−055.83 2.00E−05 1.70E−05 5.30E−05 1.70E−05 1.70E−05 3.00E−06 2.20E−05−4.30E−05 −4.00E−06 3.60E−05 5.85 1.80E−05 2.50E−05 7.30E−05 2.00E−052.30E−05 −1.00E−06 2.00E−05 −3.60E−05 −8.00E−06 5.00E−05 5.87 6.00E−062.20E−05 7.30E−05 1.80E−05 −1.00E−06 1.10E−05 8.00E−06 −3.00E−054.00E−06 2.80E−05 5.88 1.00E−05 2.70E−05 5.50E−05 1.80E−05 1.40E−051.50E−05 1.20E−05 −3.30E−05 0.00E+00 4.10E−05 5.90 1.00E−06 4.30E−056.40E−05 6.00E−06 1.00E−06 7.00E−06 7.00E−06 −4.50E−05 2.00E−06 4.20E−055.92 2.10E−05 2.70E−05 6.30E−05 2.70E−05 7.00E−06 −1.00E−06 2.40E−05−3.80E−05 −1.00E−06 5.70E−05 5.93 2.00E−06 3.80E−05 7.50E−05 1.80E−051.40E−05 5.00E−06 7.00E−06 −3.10E−05 −5.00E−06 3.30E−05 5.95 3.10E−053.40E−05 8.00E−05 1.50E−05 1.70E−05 1.00E−06 1.30E−05 −2.40E−05−6.00E−06 4.00E−05 5.97 1.00E−05 3.20E−05 6.90E−05 6.00E−06 1.20E−056.00E−06 1.20E−05 −3.50E−05 1.00E−06 4.50E−05 5.98 3.30E−05 4.70E−056.30E−05 3.10E−05 0.00E+00 1.00E−06 1.50E−05 −4.20E−05 8.00E−06 4.40E−056.00 1.30E−05 4.40E−05 7.00E−05 2.00E−05 4.00E−06 4.00E−06 −1.00E−06−2.70E−05 −1.60E−05 4.30E−05 6.02 2.30E−05 3.50E−05 6.50E−05 2.50E−051.60E−05 −7.00E−06 1.20E−05 −2.30E−05 1.50E−05 3.10E−05 6.03 1.50E−055.00E−05 6.50E−05 1.20E−05 1.00E−05 1.10E−05 6.00E−06 −1.40E−05−1.00E−06 4.10E−05 6.05 1.80E−05 3.00E−05 7.00E−05 2.00E−05 7.00E−067.00E−06 −6.00E−06 −2.10E−05 2.00E−05 2.80E−05 6.07 2.10E−05 3.50E−057.60E−05 1.70E−05 4.00E−06 1.30E−05 6.00E−06 −2.70E−05 −4.00E−064.60E−05 6.08 1.50E−05 3.20E−05 7.70E−05 2.50E−05 −4.00E−06 4.00E−061.30E−05 −3.50E−05 −1.40E−05 5.20E−05 6.10 1.60E−05 2.50E−05 7.20E−053.60E−05 1.80E−05 7.00E−06 1.10E−05 −2.60E−05 −1.60E−05 4.50E−05 6.121.70E−05 5.70E−05 7.60E−05 6.00E−06 1.00E−06 6.00E−06 6.00E−06 −2.20E−05−1.80E−05 4.40E−05 6.13 1.20E−05 3.40E−05 6.90E−05 1.70E−05 −1.10E−053.00E−06 4.00E−06 −2.50E−05 −1.20E−05 6.00E−05 6.15 1.60E−05 3.60E−056.70E−05 1.50E−05 −1.00E−06 −2.00E−06 6.00E−06 −2.60E−05 −2.00E−063.50E−05 6.17 4.20E−05 3.10E−05 7.10E−05 1.20E−05 1.40E−05 3.00E−061.10E−05 −1.80E−05 −1.00E−05 4.80E−05 6.18 2.00E−05 3.00E−05 8.70E−051.40E−05 1.20E−05 −3.00E−06 7.00E−06 −1.10E−05 −1.00E−05 4.10E−05 6.20−7.00E−06 3.20E−05 8.00E−05 1.60E−05 −4.00E−06 −7.00E−06 1.80E−05−1.30E−05 −8.00E−06 4.10E−05 6.22 1.60E−05 2.30E−05 5.40E−05 1.30E−051.00E−06 6.00E−06 3.00E−06 −1.60E−05 7.00E−06 5.20E−05 6.23 1.60E−054.00E−05 8.00E−05 2.40E−05 3.00E−06 4.00E−06 5.00E−06 −1.00E−05−1.00E−05 3.50E−05 6.25 1.80E−05 4.00E−05 4.80E−05 7.00E−06 1.20E−051.00E−06 7.00E−06 −1.80E−05 −6.00E−06 4.30E−05 6.27 2.10E−05 4.40E−057.40E−05 1.80E−05 −1.10E−05 −3.00E−06 −6.00E−06 −2.40E−05 −2.00E−064.10E−05 6.28 2.00E−05 3.30E−05 6.60E−05 1.70E−05 2.20E−05 −3.00E−062.30E−05 −8.00E−06 −3.00E−06 2.70E−05 6.30 1.70E−05 3.20E−05 8.30E−051.30E−05 1.00E−05 4.00E−06 0.00E+00 −1.40E−05 −1.00E−05 4.60E−05 6.326.00E−06 4.50E−05 7.10E−05 1.60E−05 3.00E−06 1.00E−05 8.00E−06 −5.00E−065.00E−06 3.40E−05 6.33 1.50E−05 3.20E−05 7.60E−05 2.60E−05 6.00E−062.00E−06 1.20E−05 −2.70E−05 −1.00E−06 2.30E−05 6.35 3.00E−05 4.30E−056.60E−05 2.30E−05 7.00E−06 −3.00E−06 1.60E−05 −2.40E−05 −8.00E−063.00E−05 6.37 2.70E−05 4.60E−05 6.20E−05 2.00E−05 1.30E−05 0.00E+008.00E−06 −1.40E−05 1.00E−06 3.40E−05 6.38 2.30E−05 4.20E−05 7.50E−051.80E−05 −2.00E−06 −2.00E−06 5.00E−06 −2.10E−05 −7.00E−06 4.40E−05 6.403.00E−06 2.50E−05 6.60E−05 2.70E−05 6.00E−06 −5.00E−06 1.40E−05−1.40E−05 −1.50E−05 4.20E−05 6.42 1.80E−05 3.40E−05 8.10E−05 4.00E−061.60E−05 −4.00E−06 −4.00E−06 −1.20E−05 −1.20E−05 3.10E−05 6.43 5.00E−064.70E−05 6.70E−05 2.50E−05 1.50E−05 1.00E−05 1.50E−05 −7.00E−06 1.00E−063.70E−05 6.45 1.20E−05 3.40E−05 7.10E−05 1.40E−05 1.80E−05 6.00E−067.00E−06 −1.30E−05 −8.00E−06 3.00E−05 6.47 2.30E−05 4.70E−05 7.30E−055.00E−06 5.00E−06 1.10E−05 2.00E−06 −1.10E−05 −1.40E−05 3.80E−05 6.482.40E−05 4.20E−05 6.90E−05 1.80E−05 6.00E−06 −2.00E−06 2.00E−06−5.00E−06 5.00E−06 3.30E−05 6.50 2.50E−05 4.50E−05 6.20E−05 1.60E−054.00E−06 8.00E−06 1.70E−05 −7.00E−06 0.00E+00 2.70E−05 6.52 2.10E−054.40E−05 7.30E−05 6.00E−06 1.80E−05 2.00E−06 7.00E−06 −1.00E−06−6.00E−06 3.20E−05 6.53 2.00E−05 4.40E−05 6.00E−05 3.30E−05 4.00E−061.40E−05 0.00E+00 −1.80E−05 1.00E−06 3.40E−05 6.55 1.60E−05 5.60E−055.20E−05 2.30E−05 7.00E−06 −5.00E−06 1.00E−05 −1.60E−05 2.00E−064.10E−05 6.57 1.10E−05 3.00E−05 7.50E−05 1.70E−05 1.00E−06 1.50E−051.00E−05 −1.70E−05 −1.00E−06 4.20E−05 6.58 2.10E−05 4.70E−05 6.50E−051.00E−05 1.70E−05 6.00E−06 6.00E−06 −1.00E−05 −7.00E−06 4.00E−05 6.602.30E−05 4.20E−05 8.10E−05 1.80E−05 1.00E−05 1.40E−05 1.00E−05 −1.30E−05−6.00E−06 3.80E−05 6.62 1.20E−05 3.60E−05 7.70E−05 2.20E−05 1.10E−05−1.00E−05 8.00E−06 −3.00E−06 3.00E−06 4.60E−05 6.63 8.00E−06 4.00E−056.20E−05 2.30E−05 −2.00E−06 3.00E−06 −5.00E−06 −4.00E−06 2.00E−063.80E−05 6.65 1.10E−05 4.20E−05 8.00E−05 2.80E−05 1.70E−05 4.00E−061.20E−05 −2.70E−05 4.00E−06 4.80E−05 6.67 1.60E−05 4.80E−05 7.60E−052.70E−05 1.00E−06 7.00E−06 1.20E−05 −7.00E−06 −1.10E−05 4.10E−05 6.682.20E−05 3.30E−05 8.50E−05 1.50E−05 1.00E−05 −3.00E−06 1.10E−05−2.70E−05 −2.00E−06 3.10E−05 6.70 1.30E−05 3.30E−05 7.00E−05 2.60E−051.10E−05 −1.00E−05 6.00E−06 −8.00E−06 2.00E−06 5.60E−05 6.72 2.00E−053.00E−05 8.10E−05 2.50E−05 1.10E−05 2.00E−06 2.40E−05 1.00E−05 7.00E−065.10E−05 6.73 2.60E−05 4.80E−05 6.70E−05 1.70E−05 1.20E−05 4.00E−061.80E−05 −2.10E−05 −2.00E−06 4.20E−05 6.75 2.70E−05 3.80E−05 7.20E−053.10E−05 1.00E−05 2.40E−05 2.00E−06 −2.00E−05 3.00E−06 4.30E−05 6.772.50E−05 4.50E−05 8.30E−05 3.10E−05 2.00E−05 5.00E−06 2.10E−05 −2.20E−05−1.20E−05 3.80E−05 6.78 2.10E−05 3.80E−05 7.70E−05 1.80E−05 1.50E−05−5.00E−06 7.00E−06 −1.00E−05 −1.10E−05 4.40E−05 6.80 1.40E−05 3.10E−056.60E−05 1.40E−05 7.00E−06 1.20E−05 6.00E−06 −1.60E−05 2.00E−06 3.50E−056.82 2.60E−05 4.10E−05 7.50E−05 8.00E−06 6.00E−06 1.10E−05 1.10E−05−1.80E−05 5.00E−06 4.80E−05 6.83 2.80E−05 3.60E−05 6.90E−05 2.20E−051.00E−05 1.80E−05 6.00E−06 −1.40E−05 −4.00E−06 5.40E−05 6.85 2.20E−053.70E−05 7.60E−05 2.30E−05 1.70E−05 2.00E−05 6.00E−06 −1.40E−05−1.30E−05 5.50E−05 6.87 3.40E−05 5.30E−05 7.70E−05 3.30E−05 1.60E−051.00E−05 1.20E−05 −2.10E−05 7.00E−06 5.50E−05 6.88 3.30E−05 3.80E−057.00E−05 1.20E−05 3.50E−05 1.40E−05 6.00E−06 −1.20E−05 −2.00E−066.20E−05 6.90 1.80E−05 4.20E−05 8.10E−05 2.20E−05 1.30E−05 1.20E−052.00E−06 −4.00E−06 5.00E−06 5.20E−05 6.92 1.60E−05 3.60E−05 7.10E−052.60E−05 1.00E−05 2.70E−05 −2.00E−06 −1.00E−05 1.60E−05 5.70E−05 6.934.00E−05 3.80E−05 7.60E−05 1.40E−05 4.00E−06 2.50E−05 −2.00E−06−1.60E−05 2.70E−05 5.70E−05 6.95 3.30E−05 4.40E−05 7.60E−05 1.60E−051.70E−05 1.50E−05 7.00E−06 −1.00E−06 −5.00E−06 5.10E−05 6.97 2.80E−053.00E−05 6.10E−05 1.70E−05 7.00E−06 2.10E−05 4.00E−06 −3.00E−06 1.30E−056.60E−05 6.98 2.50E−05 3.20E−05 7.40E−05 2.80E−05 1.80E−05 3.40E−056.00E−06 −1.00E−06 8.00E−06 4.30E−05 7.00 3.30E−05 3.80E−05 7.90E−052.70E−05 1.40E−05 2.30E−05 3.00E−06 0.00E+00 1.30E−05 6.50E−05 7.023.60E−05 5.30E−05 9.40E−05 6.00E−05 1.30E−05 1.80E−05 1.10E−05 −2.00E−061.10E−05 6.60E−05 7.03 3.50E−05 4.10E−05 7.50E−05 2.70E−05 1.70E−053.00E−05 1.00E−05 −1.00E−05 1.00E−05 6.20E−05 7.05 3.20E−05 3.70E−057.60E−05 3.20E−05 1.70E−05 3.10E−05 1.70E−05 −7.00E−06 1.30E−05 5.50E−057.07 3.70E−05 4.60E−05 7.50E−05 3.20E−05 1.10E−05 2.30E−05 4.00E−06−1.10E−05 1.40E−05 5.40E−05 7.08 3.20E−05 4.20E−05 8.50E−05 3.30E−051.70E−05 5.00E−05 −1.00E−06 7.00E−06 4.00E−05 5.80E−05 7.10 2.70E−054.00E−05 8.40E−05 3.80E−05 2.10E−05 4.70E−05 −4.00E−06 −2.20E−051.70E−05 6.20E−05 7.12 3.30E−05 3.80E−05 7.00E−05 4.70E−05 5.00E−064.20E−05 2.00E−06 −7.00E−06 1.80E−05 6.30E−05 7.13 1.80E−05 3.80E−057.70E−05 4.40E−05 1.60E−05 5.50E−05 −6.00E−06 −5.00E−06 1.50E−056.20E−05 7.15 4.40E−05 4.80E−05 7.70E−05 4.20E−05 2.30E−05 4.70E−05−2.00E−06 −4.00E−06 2.20E−05 7.20E−05 7.17 5.40E−05 5.60E−05 8.20E−054.50E−05 3.20E−05 5.40E−05 1.20E−05 −1.30E−05 4.30E−05 7.00E−05 7.182.50E−05 5.00E−05 7.60E−05 5.80E−05 2.40E−05 4.60E−05 1.40E−05 −1.40E−055.10E−05 7.00E−05 7.20 3.40E−05 3.60E−05 7.90E−05 5.30E−05 1.00E−056.90E−05 1.20E−05 −1.00E−05 2.80E−05 8.70E−05 7.22 3.00E−05 5.10E−057.60E−05 5.30E−05 3.10E−05 7.50E−05 −2.00E−06 −1.00E−06 3.30E−059.30E−05 7.23 2.50E−05 5.80E−05 7.60E−05 7.30E−05 3.70E−05 8.50E−05−7.00E−06 −1.30E−05 5.00E−05 9.00E−05 7.25 3.50E−05 5.70E−05 8.00E−059.40E−05 1.50E−05 9.60E−05 0.00E+00 1.10E−05 5.60E−05 8.00E−05 7.272.30E−05 7.00E−05 9.00E−05 9.30E−05 2.00E−05 9.40E−05 2.10E−05 4.00E−065.40E−05 8.70E−05 7.28 3.60E−05 6.20E−05 8.00E−05 8.00E−05 3.20E−051.31E−04 8.00E−06 −2.00E−06 5.80E−05 1.14E−04 7.30 5.30E−05 7.00E−051.01E−04 1.01E−04 2.10E−05 1.21E−04 −3.00E−06 2.40E−05 6.90E−05 1.13E−047.32 4.30E−05 6.50E−05 7.90E−05 1.06E−04 2.20E−05 1.39E−04 −1.10E−052.00E−05 8.20E−05 1.19E−04 7.33 5.00E−05 7.50E−05 8.60E−05 1.44E−043.10E−05 1.44E−04 1.20E−05 6.00E−06 8.60E−05 1.15E−04 7.35 4.10E−058.20E−05 7.10E−05 1.31E−04 2.50E−05 1.69E−04 1.50E−05 2.50E−05 9.10E−051.26E−04 7.37 4.10E−05 7.10E−05 8.50E−05 1.51E−04 2.60E−05 1.98E−04−3.00E−06 2.20E−05 1.15E−04 1.35E−04 7.38 3.70E−05 8.30E−05 8.50E−051.74E−04 1.70E−05 2.11E−04 2.00E−06 3.50E−05 1.23E−04 1.59E−04 7.404.80E−05 9.50E−05 8.30E−05 1.92E−04 1.80E−05 2.24E−04 1.40E−05 1.50E−051.35E−04 1.59E−04 7.42 5.50E−05 1.04E−04 9.40E−05 2.07E−04 3.30E−052.59E−04 1.20E−05 3.10E−05 1.62E−04 1.76E−04 7.43 3.30E−05 1.11E−049.10E−05 2.40E−04 1.70E−05 2.82E−04 1.00E−05 2.30E−05 1.74E−04 1.92E−047.45 2.30E−05 1.14E−04 9.90E−05 2.48E−04 2.80E−05 3.18E−04 1.50E−055.00E−05 1.86E−04 2.02E−04 7.47 2.70E−05 1.31E−04 9.20E−05 2.63E−042.40E−05 3.33E−04 1.50E−05 5.10E−05 2.12E−04 2.34E−04 7.48 4.60E−051.45E−04 1.10E−04 3.00E−04 2.80E−05 3.75E−04 1.00E−05 5.50E−05 2.29E−042.41E−04 7.50 4.00E−05 1.46E−04 8.60E−05 3.46E−04 3.20E−05 4.00E−047.00E−06 5.40E−05 2.43E−04 2.64E−04 7.52 4.30E−05 1.64E−04 8.60E−053.68E−04 2.20E−05 4.37E−04 8.00E−06 8.40E−05 2.64E−04 2.92E−04 7.535.10E−05 1.79E−04 8.20E−05 3.80E−04 1.80E−05 4.73E−04 8.00E−06 9.20E−053.12E−04 3.18E−04 7.55 4.80E−05 1.86E−04 9.70E−05 4.24E−04 3.10E−055.15E−04 1.30E−05 1.09E−04 3.25E−04 3.57E−04 7.57 3.70E−05 1.93E−041.01E−04 4.75E−04 1.70E−05 5.44E−04 3.40E−05 1.10E−04 3.69E−04 3.79E−047.58 3.60E−05 2.21E−04 1.16E−04 5.29E−04 3.50E−05 6.16E−04 2.80E−051.29E−04 3.94E−04 4.00E−04 7.60 4.40E−05 2.39E−04 1.04E−04 5.69E−043.40E−05 6.60E−04 2.30E−05 1.69E−04 4.26E−04 4.35E−04 7.62 5.20E−052.62E−04 1.17E−04 6.03E−04 5.00E−06 7.52E−04 2.10E−05 1.68E−04 4.74E−044.70E−04 7.63 6.30E−05 2.82E−04 1.05E−04 6.71E−04 3.20E−05 8.04E−041.60E−05 1.83E−04 5.19E−04 5.18E−04 7.65 2.60E−05 3.06E−04 1.07E−047.42E−04 3.00E−05 8.93E−04 4.70E−05 1.99E−04 5.94E−04 5.65E−04 7.673.10E−05 3.25E−04 1.15E−04 8.13E−04 2.20E−05 9.87E−04 2.60E−05 2.28E−046.40E−04 6.02E−04 7.68 4.10E−05 3.45E−04 1.22E−04 8.99E−04 2.40E−051.10E−03 3.20E−05 2.71E−04 7.25E−04 6.81E−04 7.70 4.80E−05 3.84E−041.11E−04 9.95E−04 3.50E−05 1.21E−03 3.50E−05 3.00E−04 8.04E−04 7.36E−047.72 4.00E−05 4.27E−04 1.39E−04 1.11E−03 2.50E−05 1.35E−03 3.60E−053.26E−04 9.01E−04 8.00E−04 7.73 3.70E−05 4.69E−04 1.31E−04 1.22E−033.10E−05 1.49E−03 3.70E−05 3.48E−04 9.98E−04 9.03E−04 7.75 4.30E−055.12E−04 1.28E−04 1.35E−03 4.50E−05 1.62E−03 3.60E−05 3.94E−04 1.12E−031.01E−03 7.77 4.50E−05 5.73E−04 1.29E−04 1.47E−03 3.20E−05 1.79E−034.30E−05 4.46E−04 1.23E−03 1.10E−03 7.78 4.30E−05 6.21E−04 1.56E−041.62E−03 4.40E−05 1.95E−03 2.80E−05 5.04E−04 1.36E−03 1.23E−03 7.804.40E−05 6.95E−04 1.43E−04 1.80E−03 3.30E−05 2.10E−03 4.30E−05 5.59E−041.50E−03 1.35E−03 7.82 5.20E−05 7.66E−04 1.58E−04 1.94E−03 4.00E−052.25E−03 6.00E−05 6.26E−04 1.63E−03 1.49E−03 7.83 4.80E−05 8.43E−041.62E−04 2.10E−03 3.10E−05 2.42E−03 6.30E−05 7.29E−04 1.78E−03 1.63E−037.85 6.00E−05 9.28E−04 1.70E−04 2.25E−03 4.60E−05 2.59E−03 7.40E−058.02E−04 1.94E−03 1.77E−03 7.87 4.00E−05 1.02E−03 1.85E−04 2.40E−035.20E−05 2.74E−03 5.60E−05 8.84E−04 2.07E−03 1.93E−03 7.88 4.40E−051.13E−03 2.02E−04 2.58E−03 4.60E−05 2.89E−03 5.30E−05 9.70E−04 2.22E−032.09E−03 7.90 5.60E−05 1.22E−03 1.93E−04 2.70E−03 6.60E−05 3.01E−037.90E−05 1.10E−03 2.35E−03 2.23E−03 7.92 5.10E−05 1.32E−03 2.30E−042.85E−03 6.90E−05 3.14E−03 8.40E−05 1.20E−03 2.47E−03 2.38E−03 7.934.40E−05 1.44E−03 2.35E−04 2.96E−03 6.50E−05 3.23E−03 9.50E−05 1.33E−032.58E−03 2.52E−03 7.95 6.00E−05 1.57E−03 2.47E−04 3.10E−03 7.00E−053.33E−03 9.50E−05 1.44E−03 2.71E−03 2.66E−03 7.97 5.40E−05 1.64E−032.62E−04 3.18E−03 1.01E−04 3.43E−03 1.04E−04 1.57E−03 2.82E−03 2.77E−037.98 5.40E−05 1.74E−03 2.92E−04 3.28E−03 1.12E−04 3.49E−03 1.26E−041.69E−03 2.92E−03 2.89E−03 8.00 6.10E−05 1.84E−03 3.10E−04 3.36E−031.22E−04 3.59E−03 1.29E−04 1.83E−03 3.00E−03 3.00E−03 8.02 5.20E−051.92E−03 3.45E−04 3.43E−03 1.42E−04 3.67E−03 1.30E−04 1.96E−03 3.11E−033.11E−03 8.03 5.00E−05 2.02E−03 3.52E−04 3.53E−03 1.68E−04 3.74E−031.52E−04 2.07E−03 3.20E−03 3.20E−03 8.05 4.80E−05 2.08E−03 3.81E−043.61E−03 2.12E−04 3.87E−03 1.73E−04 2.16E−03 3.29E−03 3.29E−03 8.075.20E−05 2.16E−03 4.22E−04 3.73E−03 2.38E−04 4.02E−03 1.75E−04 2.28E−033.42E−03 3.38E−03 8.08 6.20E−05 2.24E−03 4.54E−04 3.87E−03 2.63E−044.19E−03 1.86E−04 2.37E−03 3.54E−03 3.48E−03 8.10 6.60E−05 2.28E−035.04E−04 3.98E−03 3.48E−04 4.42E−03 2.27E−04 2.48E−03 3.73E−03 3.59E−038.12 4.00E−05 2.36E−03 5.34E−04 4.16E−03 4.08E−04 4.69E−03 2.34E−042.56E−03 3.91E−03 3.73E−03 8.13 6.30E−05 2.40E−03 5.76E−04 4.41E−035.00E−04 5.03E−03 2.50E−04 2.66E−03 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1.55E−03 3.09E−03 1.53E−031.16E−03 1.35E−03 11.68 2.30E−02 1.68E−03 2.64E−03 1.33E−03 1.28E−031.60E−03 3.08E−03 1.52E−03 1.15E−03 1.36E−03 11.70 2.44E−02 1.75E−032.68E−03 1.32E−03 1.24E−03 1.66E−03 3.01E−03 1.52E−03 1.12E−03 1.36E−0311.72 2.63E−02 1.80E−03 2.72E−03 1.35E−03 1.24E−03 1.72E−03 2.96E−031.50E−03 1.10E−03 1.36E−03 11.73 2.92E−02 1.88E−03 2.76E−03 1.35E−031.23E−03 1.77E−03 2.90E−03 1.48E−03 1.09E−03 1.35E−03 11.75 3.40E−021.91E−03 2.78E−03 1.36E−03 1.21E−03 1.79E−03 2.82E−03 1.44E−03 1.05E−031.35E−03 11.77 4.19E−02 1.92E−03 2.81E−03 1.34E−03 1.20E−03 1.80E−032.73E−03 1.43E−03 1.04E−03 1.33E−03 11.78 5.41E−02 1.90E−03 2.79E−031.33E−03 1.17E−03 1.79E−03 2.64E−03 1.38E−03 1.01E−03 1.31E−03 11.807.29E−02 1.87E−03 2.73E−03 1.31E−03 1.13E−03 1.74E−03 2.51E−03 1.33E−039.67E−04 1.26E−03 11.82 9.97E−02 1.80E−03 2.66E−03 1.26E−03 1.09E−031.68E−03 2.42E−03 1.29E−03 9.61E−04 1.23E−03 11.83 1.36E−01 1.71E−032.57E−03 1.24E−03 1.05E−03 1.61E−03 2.31E−03 1.23E−03 9.10E−04 1.18E−0311.85 1.81E−01 1.61E−03 2.43E−03 1.17E−03 1.03E−03 1.51E−03 2.21E−031.17E−03 8.75E−04 1.14E−03 11.87 2.34E−01 1.48E−03 2.31E−03 1.13E−039.87E−04 1.42E−03 2.12E−03 1.13E−03 8.54E−04 1.09E−03 11.88 2.94E−011.37E−03 2.18E−03 1.07E−03 9.50E−04 1.32E−03 2.03E−03 1.08E−03 8.39E−041.04E−03 11.90 3.55E−01 1.28E−03 2.03E−03 1.03E−03 8.93E−04 1.22E−031.95E−03 1.05E−03 8.43E−04 1.01E−03 11.92 4.11E−01 1.18E−03 1.91E−039.81E−04 8.73E−04 1.15E−03 1.87E−03 1.01E−03 7.94E−04 9.87E−04 11.934.57E−01 1.09E−03 1.80E−03 9.33E−04 8.72E−04 1.09E−03 1.83E−03 9.66E−047.86E−04 9.47E−04 11.95 4.88E−01 1.02E−03 1.70E−03 8.88E−04 8.44E−041.03E−03 1.74E−03 9.40E−04 7.79E−04 9.16E−04 11.97 5.02E−01 9.48E−041.62E−03 8.68E−04 8.29E−04 9.85E−04 1.69E−03 9.11E−04 7.62E−04 8.96E−0411.98 4.96E−01 8.99E−04 1.54E−03 8.37E−04 8.11E−04 9.34E−04 1.65E−038.93E−04 7.50E−04 8.88E−04 12.00 4.73E−01 8.71E−04 1.46E−03 8.22E−048.00E−04 9.07E−04 1.60E−03 8.77E−04 7.27E−04 8.65E−04 12.02 4.37E−018.32E−04 1.40E−03 7.96E−04 7.76E−04 8.98E−04 1.54E−03 8.30E−04 7.12E−048.43E−04 12.03 3.91E−01 7.88E−04 1.34E−03 7.82E−04 7.71E−04 8.54E−041.51E−03 8.18E−04 7.04E−04 8.30E−04 12.05 3.40E−01 7.63E−04 1.31E−037.70E−04 7.47E−04 8.29E−04 1.47E−03 8.06E−04 6.91E−04 8.23E−04 12.072.89E−01 7.49E−04 1.27E−03 7.45E−04 7.42E−04 8.25E−04 1.43E−03 7.78E−046.77E−04 8.08E−04 12.08 2.42E−01 7.05E−04 1.22E−03 7.42E−04 7.17E−048.32E−04 1.37E−03 7.73E−04 6.63E−04 7.96E−04 12.10 2.00E−01 7.00E−041.18E−03 7.32E−04 6.98E−04 7.89E−04 1.35E−03 7.50E−04 6.42E−04 7.55E−0412.12 1.64E−01 6.83E−04 1.16E−03 7.12E−04 6.98E−04 7.74E−04 1.31E−037.51E−04 6.43E−04 7.61E−04 12.13 1.33E−01 6.62E−04 1.14E−03 7.00E−046.78E−04 7.45E−04 1.28E−03 7.22E−04 6.29E−04 7.47E−04 12.15 1.09E−016.55E−04 1.12E−03 6.73E−04 6.58E−04 7.26E−04 1.23E−03 7.14E−04 6.08E−047.29E−04 12.17 9.03E−02 6.27E−04 1.07E−03 6.50E−04 6.44E−04 7.09E−041.21E−03 6.84E−04 6.03E−04 7.07E−04 12.18 7.53E−02 6.11E−04 1.05E−036.42E−04 6.40E−04 6.85E−04 1.17E−03 6.77E−04 5.84E−04 6.95E−04 12.206.33E−02 5.86E−04 1.02E−03 6.19E−04 6.26E−04 6.74E−04 1.14E−03 6.70E−045.89E−04 6.92E−04 12.22 5.38E−02 5.96E−04 1.01E−03 6.09E−04 6.14E−046.47E−04 1.12E−03 6.42E−04 5.82E−04 6.76E−04 12.23 4.63E−02 5.75E−049.96E−04 5.95E−04 6.05E−04 6.54E−04 1.09E−03 6.42E−04 5.58E−04 6.48E−0412.25 4.03E−02 5.82E−04 9.61E−04 5.84E−04 5.97E−04 6.26E−04 1.07E−036.28E−04 5.38E−04 6.46E−04 12.27 3.54E−02 5.48E−04 9.48E−04 5.66E−045.96E−04 6.17E−04 1.04E−03 6.19E−04 5.48E−04 6.29E−04 12.28 3.12E−025.46E−04 9.21E−04 5.86E−04 5.69E−04 6.06E−04 1.03E−03 6.23E−04 5.33E−046.36E−04 12.30 2.79E−02 5.29E−04 9.29E−04 5.48E−04 5.78E−04 5.94E−041.00E−03 5.97E−04 5.47E−04 6.26E−04 12.32 2.50E−02 5.26E−04 8.78E−045.38E−04 5.73E−04 5.82E−04 9.77E−04 5.94E−04 5.37E−04 6.26E−04 12.332.26E−02 5.14E−04 8.58E−04 5.26E−04 5.63E−04 5.81E−04 9.56E−04 5.86E−045.30E−04 6.15E−04 12.35 2.05E−02 5.06E−04 8.44E−04 5.37E−04 5.52E−045.64E−04 9.34E−04 5.58E−04 5.05E−04 6.13E−04 12.37 1.87E−02 5.01E−048.27E−04 5.28E−04 5.55E−04 5.53E−04 9.18E−04 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6.85E−04 4.79E−04 4.81E−04 4.98E−04 7.54E−04 4.83E−04 4.75E−045.12E−04 12.57 9.23E−03 4.07E−04 6.95E−04 4.56E−04 4.75E−04 4.79E−047.30E−04 4.74E−04 4.51E−04 5.19E−04 12.58 9.00E−03 4.10E−04 6.95E−044.54E−04 4.61E−04 4.69E−04 7.34E−04 4.89E−04 4.46E−04 5.18E−04 12.608.86E−03 4.09E−04 6.94E−04 4.55E−04 4.71E−04 4.71E−04 7.35E−04 4.81E−044.58E−04 5.16E−04 12.62 8.75E−03 4.18E−04 6.96E−04 4.65E−04 4.76E−044.71E−04 7.30E−04 4.70E−04 4.50E−04 5.07E−04 12.63 8.66E−03 4.32E−046.73E−04 4.50E−04 5.05E−04 4.81E−04 7.42E−04 4.75E−04 4.67E−04 5.14E−0412.65 8.57E−03 4.30E−04 6.80E−04 4.90E−04 5.35E−04 4.99E−04 7.52E−044.68E−04 4.79E−04 5.15E−04 12.67 8.45E−03 4.48E−04 6.86E−04 4.95E−045.74E−04 5.08E−04 7.45E−04 4.84E−04 4.81E−04 5.24E−04 12.68 8.32E−034.75E−04 6.66E−04 5.36E−04 6.29E−04 5.30E−04 7.75E−04 4.81E−04 4.96E−045.19E−04 12.70 8.18E−03 4.90E−04 6.60E−04 6.02E−04 6.98E−04 5.95E−048.27E−04 5.07E−04 5.30E−04 5.27E−04 12.72 7.99E−03 5.04E−04 6.47E−046.74E−04 8.18E−04 6.28E−04 8.83E−04 5.39E−04 5.69E−04 5.24E−04 12.737.81E−03 5.40E−04 6.51E−04 7.56E−04 9.43E−04 7.43E−04 9.81E−04 5.86E−046.21E−04 5.48E−04 12.75 7.62E−03 5.92E−04 6.46E−04 8.87E−04 1.13E−038.59E−04 1.09E−03 6.54E−04 7.01E−04 5.59E−04 12.77 7.41E−03 6.31E−046.62E−04 1.08E−03 1.36E−03 1.01E−03 1.18E−03 7.36E−04 7.65E−04 5.84E−0412.78 7.25E−03 7.09E−04 6.93E−04 1.31E−03 1.58E−03 1.21E−03 1.26E−038.28E−04 8.28E−04 6.17E−04 12.80 7.09E−03 8.28E−04 7.25E−04 1.60E−031.78E−03 1.42E−03 1.32E−03 9.03E−04 8.75E−04 6.71E−04 12.82 6.92E−039.60E−04 7.52E−04 1.86E−03 1.88E−03 1.57E−03 1.32E−03 9.68E−04 8.98E−047.22E−04 12.83 6.74E−03 1.11E−03 7.75E−04 2.09E−03 1.91E−03 1.67E−031.31E−03 1.00E−03 9.29E−04 7.72E−04 12.85 6.57E−03 1.29E−03 8.22E−042.21E−03 1.81E−03 1.67E−03 1.27E−03 1.01E−03 9.01E−04 8.45E−04 12.876.41E−03 1.44E−03 8.53E−04 2.21E−03 1.68E−03 1.62E−03 1.22E−03 1.01E−038.54E−04 8.67E−04 12.88 6.25E−03 1.60E−03 8.67E−04 2.08E−03 1.50E−031.48E−03 1.13E−03 9.89E−04 8.11E−04 8.84E−04 12.90 6.08E−03 1.68E−038.64E−04 1.89E−03 1.29E−03 1.31E−03 1.05E−03 9.43E−04 7.65E−04 8.94E−0412.92 5.89E−03 1.72E−03 8.62E−04 1.64E−03 1.14E−03 1.14E−03 9.62E−048.68E−04 7.10E−04 8.51E−04 12.93 5.72E−03 1.66E−03 8.22E−04 1.40E−039.86E−04 9.82E−04 8.67E−04 7.85E−04 6.31E−04 8.13E−04 12.95 5.56E−031.55E−03 7.74E−04 1.18E−03 8.51E−04 8.54E−04 8.00E−04 7.16E−04 5.95E−047.69E−04 12.97 5.41E−03 1.39E−03 7.31E−04 1.00E−03 7.71E−04 7.56E−047.19E−04 6.35E−04 5.44E−04 7.11E−04 12.98 5.21E−03 1.21E−03 6.86E−048.58E−04 6.95E−04 6.82E−04 6.71E−04 5.96E−04 5.19E−04 6.46E−04 13.005.06E−03 1.04E−03 6.38E−04 7.49E−04 6.29E−04 6.28E−04 6.07E−04 5.18E−044.79E−04 5.98E−04 13.02 4.94E−03 8.82E−04 5.93E−04 6.71E−04 5.79E−045.77E−04 5.77E−04 4.89E−04 4.50E−04 5.70E−04 13.03 4.81E−03 7.54E−045.60E−04 6.28E−04 5.78E−04 5.42E−04 5.47E−04 4.44E−04 4.42E−04 5.44E−0413.05 4.67E−03 6.65E−04 5.32E−04 5.76E−04 5.35E−04 5.22E−04 5.25E−044.00E−04 4.20E−04 4.90E−04 13.07 4.52E−03 5.75E−04 5.12E−04 5.58E−045.06E−04 4.90E−04 5.08E−04 3.95E−04 3.98E−04 4.84E−04 13.08 4.41E−035.03E−04 5.00E−04 5.13E−04 5.06E−04 4.78E−04 4.88E−04 3.77E−04 3.75E−044.85E−04 13.10 4.30E−03 4.68E−04 4.68E−04 4.88E−04 4.91E−04 4.63E−044.80E−04 3.68E−04 3.57E−04 4.59E−04 13.12 4.21E−03 4.36E−04 4.67E−044.58E−04 4.54E−04 4.40E−04 4.42E−04 3.42E−04 3.50E−04 4.45E−04 13.134.14E−03 4.05E−04 4.40E−04 4.49E−04 4.60E−04 4.38E−04 4.30E−04 3.42E−043.48E−04 4.36E−04 13.15 3.99E−03 3.86E−04 4.32E−04 4.30E−04 4.41E−044.01E−04 4.20E−04 3.14E−04 3.36E−04 4.32E−04 13.17 3.91E−03 3.55E−044.28E−04 4.26E−04 4.29E−04 3.96E−04 4.18E−04 3.13E−04 3.36E−04 3.98E−0413.18 3.83E−03 3.38E−04 4.15E−04 4.02E−04 4.27E−04 3.91E−04 4.00E−042.91E−04 3.20E−04 4.16E−04 13.20 3.76E−03 3.40E−04 4.25E−04 3.97E−044.28E−04 3.89E−04 4.06E−04 2.90E−04 3.22E−04 4.07E−04 13.22 3.66E−033.19E−04 4.10E−04 3.89E−04 4.21E−04 3.97E−04 3.88E−04 2.91E−04 3.11E−044.05E−04 13.23 3.57E−03 3.04E−04 4.17E−04 3.72E−04 4.22E−04 3.68E−043.86E−04 2.86E−04 3.09E−04 3.96E−04 13.25 3.47E−03 3.00E−04 3.98E−043.87E−04 4.10E−04 3.72E−04 3.73E−04 2.73E−04 3.12E−04 3.96E−04 13.273.41E−03 2.92E−04 3.88E−04 3.55E−04 4.11E−04 3.68E−04 3.77E−04 2.63E−042.92E−04 3.89E−04 13.28 3.34E−03 2.93E−04 3.75E−04 3.55E−04 4.15E−043.59E−04 3.58E−04 2.69E−04 3.06E−04 4.01E−04 13.30 3.24E−03 3.00E−043.72E−04 3.67E−04 3.94E−04 3.55E−04 3.70E−04 2.66E−04 3.02E−04 3.97E−0413.32 3.21E−03 2.76E−04 3.68E−04 3.65E−04 3.95E−04 3.67E−04 3.51E−042.54E−04 2.84E−04 3.92E−04 13.33 3.11E−03 2.67E−04 3.78E−04 3.47E−043.72E−04 3.46E−04 3.47E−04 2.48E−04 3.03E−04 3.98E−04 13.35 3.04E−032.67E−04 3.63E−04 3.55E−04 3.76E−04 3.29E−04 3.36E−04 2.45E−04 2.90E−043.73E−04 13.37 2.97E−03 2.68E−04 3.61E−04 3.20E−04 3.58E−04 3.21E−043.32E−04 2.43E−04 2.76E−04 3.82E−04 13.38 2.88E−03 2.77E−04 3.50E−043.30E−04 3.49E−04 3.70E−04 3.13E−04 2.21E−04 2.69E−04 3.71E−04 13.402.80E−03 2.62E−04 3.42E−04 3.11E−04 3.52E−04 3.26E−04 3.22E−04 2.20E−042.77E−04 3.77E−04 13.42 2.73E−03 2.52E−04 3.53E−04 2.99E−04 3.31E−043.08E−04 3.17E−04 2.18E−04 2.78E−04 3.66E−04 13.43 2.68E−03 2.39E−043.38E−04 3.10E−04 3.47E−04 3.12E−04 3.03E−04 2.28E−04 2.77E−04 3.63E−0413.45 2.59E−03 2.41E−04 3.36E−04 2.84E−04 3.38E−04 2.92E−04 3.08E−042.14E−04 2.56E−04 3.65E−04 13.47 2.50E−03 2.47E−04 3.35E−04 2.88E−043.19E−04 3.08E−04 3.03E−04 2.14E−04 2.58E−04 3.65E−04 13.48 2.43E−032.47E−04 3.25E−04 2.98E−04 3.27E−04 2.94E−04 3.03E−04 2.00E−04 2.59E−043.70E−04 13.50 2.37E−03 2.42E−04 3.03E−04 2.82E−04 3.18E−04 2.94E−042.97E−04 2.01E−04 2.63E−04 3.49E−04 13.52 2.29E−03 2.47E−04 2.96E−042.81E−04 3.29E−04 2.82E−04 3.03E−04 2.01E−04 2.58E−04 3.58E−04 13.532.23E−03 2.45E−04 3.01E−04 2.71E−04 3.18E−04 2.79E−04 2.82E−04 1.92E−042.62E−04 3.56E−04 13.55 2.14E−03 2.30E−04 3.03E−04 2.86E−04 3.28E−042.78E−04 2.76E−04 1.89E−04 2.45E−04 3.52E−04 13.57 2.07E−03 2.25E−043.11E−04 2.70E−04 2.96E−04 2.87E−04 2.87E−04 1.75E−04 2.45E−04 3.59E−0413.58 2.01E−03 2.37E−04 3.03E−04 2.69E−04 3.17E−04 2.79E−04 2.66E−041.81E−04 2.54E−04 3.42E−04 13.60 1.94E−03 2.27E−04 3.00E−04 2.81E−043.16E−04 2.82E−04 2.63E−04 1.82E−04 2.56E−04 3.50E−04 13.62 1.86E−032.14E−04 3.10E−04 2.76E−04 2.92E−04 2.86E−04 2.87E−04 1.68E−04 2.53E−043.40E−04 13.63 1.80E−03 2.25E−04 2.77E−04 2.63E−04 2.89E−04 2.71E−042.49E−04 1.91E−04 2.57E−04 3.53E−04 13.65 1.75E−03 2.21E−04 2.87E−042.68E−04 2.98E−04 2.83E−04 2.59E−04 1.71E−04 2.57E−04 3.27E−04 13.671.68E−03 2.29E−04 2.80E−04 2.43E−04 3.02E−04 2.74E−04 2.53E−04 1.71E−042.44E−04 3.26E−04 13.68 1.65E−03 2.37E−04 2.83E−04 2.50E−04 2.98E−042.79E−04 2.41E−04 1.53E−04 2.57E−04 3.32E−04 13.70 1.60E−03 2.17E−042.73E−04 2.41E−04 2.91E−04 2.60E−04 2.35E−04 1.62E−04 2.43E−04 3.31E−0413.72 1.54E−03 2.07E−04 2.60E−04 2.52E−04 2.82E−04 2.66E−04 2.37E−041.65E−04 2.31E−04 3.25E−04 13.73 1.47E−03 2.03E−04 2.67E−04 2.43E−042.91E−04 2.82E−04 2.32E−04 1.62E−04 2.58E−04 3.26E−04 13.75 1.43E−032.12E−04 2.64E−04 2.43E−04 2.91E−04 2.58E−04 2.27E−04 1.66E−04 2.37E−043.22E−04 13.77 1.39E−03 2.05E−04 2.59E−04 2.30E−04 2.81E−04 2.82E−042.20E−04 1.62E−04 2.41E−04 3.26E−04 13.78 1.33E−03 2.18E−04 2.63E−042.25E−04 2.73E−04 2.50E−04 2.17E−04 1.46E−04 2.32E−04 3.12E−04 13.801.30E−03 2.13E−04 2.49E−04 2.30E−04 2.82E−04 2.59E−04 2.28E−04 1.59E−042.42E−04 3.27E−04 13.82 1.25E−03 2.09E−04 2.42E−04 2.35E−04 2.72E−042.54E−04 2.24E−04 1.60E−04 2.32E−04 3.13E−04 13.83 1.25E−03 2.11E−042.50E−04 2.34E−04 2.70E−04 2.66E−04 2.05E−04 1.42E−04 2.34E−04 3.11E−0413.85 1.20E−03 1.98E−04 2.44E−04 2.24E−04 2.53E−04 2.59E−04 2.02E−041.40E−04 2.30E−04 3.20E−04 13.87 1.14E−03 2.03E−04 2.34E−04 2.25E−042.61E−04 2.61E−04 2.07E−04 1.52E−04 2.29E−04 3.31E−04 13.88 1.10E−031.89E−04 2.30E−04 2.22E−04 2.72E−04 2.63E−04 2.01E−04 1.45E−04 2.30E−043.10E−04 13.90 1.07E−03 1.97E−04 2.39E−04 2.11E−04 2.61E−04 2.49E−042.03E−04 1.45E−04 2.28E−04 3.02E−04 13.92 1.04E−03 1.99E−04 2.23E−042.09E−04 2.51E−04 2.45E−04 1.86E−04 1.46E−04 2.25E−04 3.22E−04 13.931.02E−03 2.00E−04 2.32E−04 2.19E−04 2.58E−04 2.38E−04 1.94E−04 1.49E−042.28E−04 3.02E−04 13.95 9.71E−04 1.90E−04 2.22E−04 2.13E−04 2.52E−042.52E−04 1.90E−04 1.38E−04 2.31E−04 3.20E−04 13.97 9.52E−04 1.80E−042.20E−04 2.02E−04 2.60E−04 2.50E−04 2.05E−04 1.39E−04 2.37E−04 3.03E−0413.98 9.27E−04 1.89E−04 2.25E−04 2.20E−04 2.61E−04 2.44E−04 1.83E−041.38E−04 2.32E−04 3.01E−04 14.00 9.02E−04 1.80E−04 2.23E−04 1.92E−042.71E−04 2.34E−04 1.83E−04 1.36E−04 2.29E−04 2.99E−04 14.02 8.91E−041.70E−04 2.20E−04 1.95E−04 2.45E−04 2.47E−04 1.80E−04 1.36E−04 2.35E−043.09E−04 14.03 8.64E−04 1.90E−04 2.18E−04 2.01E−04 2.51E−04 2.29E−041.75E−04 1.19E−04 2.27E−04 2.96E−04 14.05 8.38E−04 1.93E−04 2.19E−042.05E−04 2.31E−04 2.42E−04 1.86E−04 1.36E−04 2.35E−04 2.99E−04 14.078.18E−04 1.73E−04 2.23E−04 2.10E−04 2.42E−04 2.35E−04 1.68E−04 1.25E−042.22E−04 3.00E−04 14.08 7.92E−04 1.81E−04 2.05E−04 2.01E−04 2.14E−042.24E−04 1.65E−04 1.21E−04 2.24E−04 3.00E−04 14.10 7.82E−04 1.69E−042.07E−04 1.94E−04 2.25E−04 2.44E−04 1.69E−04 1.32E−04 2.35E−04 3.02E−0414.12 7.61E−04 1.86E−04 2.02E−04 1.90E−04 2.37E−04 2.47E−04 1.81E−041.26E−04 2.49E−04 2.93E−04 14.13 7.49E−04 1.78E−04 2.22E−04 1.86E−042.35E−04 2.23E−04 1.81E−04 1.15E−04 2.23E−04 2.93E−04 14.15 7.32E−041.73E−04 2.03E−04 2.00E−04 2.42E−04 2.42E−04 1.83E−04 1.23E−04 2.21E−042.97E−04 14.17 7.13E−04 1.66E−04 2.00E−04 1.82E−04 2.28E−04 2.28E−041.69E−04 1.15E−04 2.21E−04 2.90E−04 14.18 7.03E−04 1.66E−04 2.07E−041.93E−04 2.39E−04 2.35E−04 1.66E−04 1.16E−04 2.37E−04 2.83E−04 14.206.75E−04 1.68E−04 2.05E−04 1.90E−04 2.32E−04 2.32E−04 1.55E−04 1.49E−042.40E−04 2.89E−04 14.22 6.70E−04 1.70E−04 2.05E−04 1.92E−04 2.33E−042.29E−04 1.69E−04 1.20E−04 2.17E−04 2.86E−04 14.23 6.54E−04 1.81E−041.86E−04 1.89E−04 2.23E−04 2.29E−04 1.69E−04 1.16E−04 2.27E−04 2.82E−0414.25 6.41E−04 1.95E−04 2.12E−04 1.84E−04 2.40E−04 2.29E−04 1.65E−041.35E−04 2.22E−04 2.89E−04 14.27 6.28E−04 1.68E−04 1.91E−04 1.83E−042.22E−04 2.30E−04 1.55E−04 1.11E−04 2.11E−04 2.81E−04 14.28 6.40E−041.75E−04 1.90E−04 1.90E−04 2.25E−04 2.42E−04 1.69E−04 1.07E−04 2.35E−042.83E−04 14.30 6.06E−04 1.72E−04 1.84E−04 1.84E−04 2.22E−04 2.37E−041.55E−04 1.25E−04 2.21E−04 2.84E−04 14.32 5.98E−04 1.81E−04 1.89E−041.76E−04 2.28E−04 2.15E−04 1.79E−04 1.07E−04 2.32E−04 2.98E−04 14.335.91E−04 1.82E−04 1.88E−04 1.82E−04 2.24E−04 2.19E−04 1.56E−04 1.05E−042.24E−04 2.81E−04 14.35 5.75E−04 1.80E−04 2.00E−04 1.73E−04 2.32E−042.25E−04 1.52E−04 1.11E−04 2.27E−04 2.92E−04 14.37 5.62E−04 1.58E−041.93E−04 1.79E−04 2.24E−04 2.21E−04 1.66E−04 1.12E−04 2.20E−04 2.69E−0414.38 5.57E−04 1.76E−04 2.02E−04 1.81E−04 2.25E−04 2.34E−04 1.61E−041.10E−04 2.25E−04 2.86E−04 14.40 5.42E−04 1.69E−04 1.83E−04 1.82E−042.20E−04 2.24E−04 1.49E−04 1.15E−04 2.29E−04 2.91E−04 14.42 5.25E−041.88E−04 1.90E−04 1.78E−04 2.37E−04 2.20E−04 1.53E−04 1.09E−04 2.21E−042.70E−04 14.43 5.26E−04 1.69E−04 1.95E−04 1.71E−04 2.10E−04 2.08E−041.59E−04 9.30E−05 2.20E−04 2.71E−04 14.45 5.26E−04 1.64E−04 1.97E−041.80E−04 2.08E−04 2.19E−04 1.53E−04 1.03E−04 2.14E−04 2.73E−04 14.475.19E−04 1.74E−04 2.00E−04 1.84E−04 2.13E−04 2.08E−04 1.71E−04 1.11E−042.35E−04 2.64E−04 14.48 5.06E−04 1.76E−04 1.78E−04 1.75E−04 2.22E−042.18E−04 1.58E−04 1.13E−04 2.23E−04 2.74E−04 14.50 4.93E−04 1.68E−041.88E−04 1.59E−04 2.02E−04 2.17E−04 1.62E−04 9.90E−05 2.31E−04 2.77E−0414.52 4.95E−04 1.63E−04 1.98E−04 1.72E−04 2.11E−04 2.05E−04 1.60E−041.05E−04 2.30E−04 3.02E−04 14.53 4.78E−04 1.60E−04 2.00E−04 1.69E−041.98E−04 2.04E−04 1.49E−04 1.01E−04 2.17E−04 2.79E−04 14.55 4.78E−041.80E−04 1.99E−04 1.98E−04 2.15E−04 2.17E−04 1.69E−04 1.03E−04 2.24E−042.74E−04 14.57 4.81E−04 1.73E−04 1.78E−04 1.59E−04 2.13E−04 1.93E−041.56E−04 1.00E−04 2.25E−04 2.63E−04 14.58 4.65E−04 1.52E−04 1.86E−041.72E−04 1.97E−04 2.09E−04 1.58E−04 9.60E−05 2.33E−04 2.73E−04 14.604.83E−04 1.58E−04 1.84E−04 1.61E−04 2.12E−04 2.03E−04 1.65E−04 9.60E−052.32E−04 2.66E−04 14.62 4.46E−04 1.48E−04 1.92E−04 1.53E−04 2.02E−042.10E−04 1.55E−04 1.17E−04 2.29E−04 2.73E−04 14.63 4.54E−04 1.72E−041.89E−04 1.63E−04 2.07E−04 2.01E−04 1.62E−04 1.03E−04 2.38E−04 2.63E−0414.65 4.41E−04 1.83E−04 1.85E−04 1.42E−04 1.99E−04 2.03E−04 1.68E−049.90E−05 2.39E−04 2.69E−04 14.67 4.37E−04 1.65E−04 1.91E−04 1.66E−042.19E−04 2.05E−04 1.79E−04 1.12E−04 2.51E−04 2.82E−04 14.68 4.35E−041.54E−04 1.95E−04 1.60E−04 2.03E−04 1.99E−04 1.66E−04 1.02E−04 2.68E−042.68E−04 14.70 4.22E−04 1.72E−04 1.86E−04 1.53E−04 2.10E−04 1.91E−041.73E−04 1.03E−04 2.60E−04 2.73E−04 14.72 4.18E−04 1.66E−04 2.10E−041.51E−04 2.21E−04 2.12E−04 1.80E−04 1.07E−04 2.53E−04 2.70E−04 14.734.24E−04 1.64E−04 1.98E−04 1.59E−04 2.17E−04 2.04E−04 1.81E−04 1.21E−042.71E−04 2.79E−04 14.75 4.18E−04 1.68E−04 2.04E−04 1.59E−04 2.20E−042.11E−04 1.85E−04 1.23E−04 2.61E−04 2.84E−04 14.77 4.54E−04 1.63E−041.89E−04 1.42E−04 2.39E−04 2.12E−04 1.81E−04 1.23E−04 2.67E−04 2.86E−0414.78 4.14E−04 1.79E−04 2.04E−04 1.62E−04 2.15E−04 2.11E−04 1.75E−041.28E−04 2.62E−04 2.80E−04 14.80 3.79E−04 1.72E−04 1.90E−04 1.60E−042.24E−04 2.10E−04 1.93E−04 1.12E−04 2.66E−04 2.77E−04 14.82 3.91E−041.82E−04 1.95E−04 1.68E−04 2.30E−04 2.09E−04 1.75E−04 1.15E−04 2.60E−042.87E−04 14.83 3.87E−04 1.74E−04 2.02E−04 1.65E−04 2.17E−04 2.05E−041.78E−04 1.11E−04 2.30E−04 2.94E−04 14.85 3.76E−04 1.76E−04 2.12E−041.65E−04 2.03E−04 2.09E−04 1.75E−04 1.12E−04 2.43E−04 2.86E−04 14.873.68E−04 1.75E−04 2.01E−04 1.53E−04 2.12E−04 2.07E−04 1.84E−04 9.90E−052.38E−04 2.64E−04 14.88 3.77E−04 1.63E−04 1.92E−04 1.70E−04 2.21E−041.98E−04 1.65E−04 1.05E−04 2.45E−04 2.66E−04 14.90 3.65E−04 1.52E−041.85E−04 1.51E−04 1.97E−04 1.99E−04 1.85E−04 1.09E−04 2.34E−04 2.61E−0414.92 3.69E−04 1.63E−04 1.99E−04 1.56E−04 1.90E−04 1.82E−04 1.89E−041.01E−04 2.42E−04 2.60E−04 14.93 3.56E−04 1.65E−04 1.80E−04 1.52E−041.97E−04 2.03E−04 1.66E−04 9.50E−05 2.25E−04 2.57E−04 14.95 3.46E−041.58E−04 1.85E−04 1.41E−04 2.07E−04 2.00E−04 1.83E−04 9.10E−05 2.48E−042.53E−04 14.97 3.39E−04 1.54E−04 1.76E−04 1.69E−04 2.00E−04 1.85E−041.90E−04 8.20E−05 2.29E−04 2.50E−04 14.98 3.47E−04 1.44E−04 1.84E−041.29E−04 1.93E−04 1.91E−04 1.86E−04 9.70E−05 2.42E−04 2.54E−04 15.003.52E−04 1.41E−04 1.82E−04 1.38E−04 1.91E−04 1.86E−04 1.82E−04 1.02E−042.30E−04 2.43E−04 15.02 3.30E−04 1.51E−04 1.81E−04 1.39E−04 1.97E−042.01E−04 1.76E−04 1.02E−04 2.32E−04 2.51E−04 15.03 3.35E−04 1.53E−041.90E−04 1.45E−04 1.92E−04 1.86E−04 1.71E−04 8.50E−05 2.32E−04 2.57E−0415.05 3.42E−04 1.40E−04 1.73E−04 1.46E−04 1.93E−04 1.91E−04 1.81E−047.60E−05 2.21E−04 2.56E−04 15.07 3.18E−04 1.34E−04 1.76E−04 1.36E−042.02E−04 1.89E−04 1.64E−04 9.10E−05 2.35E−04 2.62E−04 15.08 3.30E−041.40E−04 1.83E−04 1.30E−04 1.94E−04 1.83E−04 1.85E−04 9.30E−05 2.37E−042.38E−04 15.10 3.27E−04 1.31E−04 1.76E−04 1.35E−04 1.85E−04 1.79E−041.74E−04 9.00E−05 2.15E−04 2.60E−04 15.12 3.12E−04 1.32E−04 1.81E−041.40E−04 1.88E−04 1.92E−04 1.88E−04 8.10E−05 2.35E−04 2.49E−04 15.133.14E−04 1.44E−04 1.89E−04 1.43E−04 1.89E−04 1.85E−04 1.65E−04 8.60E−052.23E−04 2.51E−04 15.15 3.13E−04 1.19E−04 1.85E−04 1.51E−04 1.85E−041.95E−04 1.74E−04 7.60E−05 2.28E−04 2.31E−04 15.17 3.02E−04 1.25E−041.85E−04 1.34E−04 1.78E−04 1.83E−04 1.79E−04 1.02E−04 2.39E−04 2.34E−0415.18 3.12E−04 1.39E−04 1.83E−04 1.28E−04 1.78E−04 1.73E−04 2.04E−048.70E−05 2.35E−04 2.39E−04 15.20 3.12E−04 1.22E−04 1.61E−04 1.24E−041.70E−04 1.85E−04 1.75E−04 8.40E−05 2.25E−04 2.57E−04 15.22 3.08E−041.23E−04 1.69E−04 1.29E−04 1.70E−04 1.65E−04 1.91E−04 8.10E−05 2.25E−042.35E−04 15.23 3.11E−04 1.29E−04 1.80E−04 1.39E−04 1.80E−04 1.74E−041.73E−04 9.10E−05 2.23E−04 2.37E−04 15.25 3.00E−04 1.26E−04 1.78E−041.35E−04 1.70E−04 1.73E−04 1.73E−04 7.40E−05 2.23E−04 2.40E−04 15.272.98E−04 1.14E−04 1.86E−04 1.29E−04 1.75E−04 1.84E−04 1.74E−04 9.10E−052.20E−04 2.69E−04 15.28 2.89E−04 1.11E−04 1.62E−04 1.09E−04 1.76E−041.88E−04 1.85E−04 7.60E−05 2.13E−04 2.25E−04 15.30 2.94E−04 1.21E−041.65E−04 1.16E−04 1.69E−04 1.79E−04 1.66E−04 8.10E−05 2.11E−04 2.41E−0415.32 2.96E−04 1.07E−04 1.71E−04 1.12E−04 1.74E−04 1.79E−04 1.72E−048.20E−05 2.20E−04 2.47E−04 15.33 2.92E−04 1.24E−04 1.62E−04 1.17E−041.59E−04 1.70E−04 1.82E−04 6.10E−05 2.03E−04 2.23E−04 15.35 2.99E−041.16E−04 1.66E−04 1.16E−04 1.68E−04 1.62E−04 1.73E−04 7.40E−05 2.24E−042.47E−04 15.37 3.07E−04 1.19E−04 1.76E−04 1.06E−04 1.72E−04 1.69E−041.76E−04 7.00E−05 2.14E−04 2.39E−04 15.38 2.99E−04 1.14E−04 1.56E−041.04E−04 1.64E−04 1.68E−04 1.69E−04 8.30E−05 2.11E−04 2.44E−04 15.403.02E−04 1.11E−04 1.75E−04 1.01E−04 1.64E−04 1.70E−04 1.62E−04 5.60E−052.17E−04 2.39E−04 15.42 2.84E−04 1.13E−04 1.75E−04 1.07E−04 1.49E−041.52E−04 1.74E−04 7.10E−05 1.93E−04 2.42E−04 15.43 2.92E−04 1.15E−041.61E−04 1.09E−04 1.74E−04 1.53E−04 1.68E−04 6.00E−05 2.03E−04 2.28E−0415.45 2.86E−04 9.50E−05 1.65E−04 1.10E−04 1.70E−04 1.65E−04 1.70E−046.40E−05 2.29E−04 2.33E−04 15.47 2.77E−04 1.13E−04 1.69E−04 1.11E−041.81E−04 1.63E−04 1.66E−04 6.20E−05 1.95E−04 2.23E−04 15.48 2.88E−041.01E−04 1.55E−04 1.11E−04 1.68E−04 1.60E−04 1.65E−04 7.30E−05 2.19E−042.38E−04 15.50 2.88E−04 9.40E−05 1.68E−04 1.25E−04 1.78E−04 1.63E−041.79E−04 7.60E−05 2.07E−04 2.24E−04 15.52 2.82E−04 1.11E−04 1.51E−041.01E−04 1.69E−04 1.53E−04 1.61E−04 7.10E−05 1.91E−04 2.34E−04 15.532.87E−04 9.60E−05 1.71E−04 1.26E−04 1.73E−04 1.50E−04 1.68E−04 5.70E−052.23E−04 2.17E−04 15.55 2.77E−04 1.09E−04 1.61E−04 9.70E−05 1.59E−041.72E−04 1.64E−04 6.70E−05 2.01E−04 2.20E−04 15.57 2.74E−04 1.05E−041.65E−04 1.06E−04 1.56E−04 1.45E−04 1.63E−04 7.00E−05 1.97E−04 2.33E−0415.58 2.81E−04 1.02E−04 1.70E−04 1.06E−04 1.59E−04 1.61E−04 1.52E−046.40E−05 1.98E−04 2.29E−04 15.60 2.70E−04 1.01E−04 1.58E−04 1.06E−041.63E−04 1.56E−04 1.52E−04 6.30E−05 2.01E−04 2.34E−04 15.62 2.81E−049.40E−05 1.61E−04 1.05E−04 1.52E−04 1.56E−04 1.75E−04 5.80E−05 2.15E−042.14E−04 15.63 2.68E−04 9.20E−05 1.65E−04 1.02E−04 1.56E−04 1.60E−041.76E−04 6.90E−05 2.02E−04 2.23E−04 15.65 2.64E−04 9.10E−05 1.51E−049.60E−05 1.69E−04 1.42E−04 1.64E−04 5.40E−05 1.98E−04 2.33E−04 15.672.64E−04 1.03E−04 1.74E−04 1.07E−04 1.58E−04 1.52E−04 1.69E−04 7.00E−051.84E−04 2.31E−04 15.68 2.63E−04 8.40E−05 1.51E−04 9.30E−05 1.53E−041.52E−04 1.64E−04 5.20E−05 1.98E−04 2.29E−04 15.70 2.70E−04 1.06E−041.54E−04 1.02E−04 1.49E−04 1.41E−04 1.65E−04 5.00E−05 1.91E−04 2.11E−0415.72 2.52E−04 8.10E−05 1.60E−04 9.30E−05 1.66E−04 1.48E−04 1.65E−046.20E−05 1.84E−04 2.33E−04 15.73 2.58E−04 9.30E−05 1.55E−04 1.04E−041.86E−04 1.51E−04 1.64E−04 5.80E−05 1.92E−04 2.20E−04 15.75 2.66E−048.90E−05 1.66E−04 9.90E−05 1.63E−04 1.45E−04 1.62E−04 6.00E−05 2.02E−042.28E−04 15.77 2.62E−04 7.50E−05 1.55E−04 9.20E−05 1.74E−04 1.39E−041.66E−04 5.50E−05 1.94E−04 2.33E−04 15.78 2.58E−04 9.60E−05 1.44E−049.90E−05 1.64E−04 1.50E−04 1.61E−04 6.00E−05 1.92E−04 2.13E−04 15.802.42E−04 7.70E−05 1.50E−04 9.40E−05 1.94E−04 1.39E−04 1.65E−04 3.50E−051.88E−04 2.32E−04 15.82 2.57E−04 9.50E−05 1.60E−04 9.20E−05 1.78E−041.45E−04 1.63E−04 5.00E−05 1.85E−04 2.21E−04 15.83 2.52E−04 8.30E−051.61E−04 1.03E−04 1.79E−04 1.53E−04 1.61E−04 4.40E−05 1.89E−04 2.24E−0415.85 2.54E−04 9.40E−05 1.50E−04 1.02E−04 1.68E−04 1.42E−04 1.66E−046.70E−05 1.84E−04 2.44E−04 15.87 2.57E−04 8.50E−05 1.53E−04 1.09E−041.72E−04 1.50E−04 1.78E−04 5.10E−05 1.94E−04 2.34E−04 15.88 2.44E−048.90E−05 1.50E−04 1.05E−04 1.83E−04 1.58E−04 1.56E−04 5.40E−05 2.09E−042.33E−04 15.90 2.54E−04 8.20E−05 1.68E−04 1.26E−04 1.78E−04 1.50E−041.58E−04 4.30E−05 1.98E−04 2.20E−04 15.92 2.42E−04 7.90E−05 1.68E−041.13E−04 1.65E−04 1.42E−04 1.53E−04 4.80E−05 1.82E−04 2.29E−04 15.932.15E−04 7.90E−05 1.54E−04 1.24E−04 1.66E−04 1.44E−04 1.64E−04 6.50E−051.79E−04 2.25E−04 15.95 2.42E−04 8.70E−05 1.54E−04 1.17E−04 1.64E−041.49E−04 1.45E−04 4.60E−05 1.92E−04 2.12E−04 15.97 2.37E−04 7.40E−051.69E−04 1.06E−04 1.65E−04 1.34E−04 1.43E−04 5.00E−05 1.88E−04 2.19E−0415.98 2.35E−04 7.00E−05 1.52E−04 1.10E−04 1.62E−04 1.45E−04 1.44E−044.50E−05 1.80E−04 2.34E−04 16.00 2.40E−04 8.90E−05 1.56E−04 1.04E−041.58E−04 1.33E−04 1.60E−04 5.30E−05 1.64E−04 2.31E−04 16.02 2.43E−047.90E−05 1.39E−04 1.12E−04 1.54E−04 1.30E−04 1.43E−04 6.20E−05 1.74E−042.15E−04 16.03 2.33E−04 6.70E−05 1.51E−04 8.00E−05 1.58E−04 1.44E−041.53E−04 4.70E−05 1.68E−04 2.21E−04 16.05 2.31E−04 8.30E−05 1.32E−048.60E−05 1.49E−04 1.25E−04 1.49E−04 3.10E−05 1.80E−04 2.14E−04 16.072.31E−04 7.10E−05 1.50E−04 1.06E−04 1.40E−04 1.44E−04 1.55E−04 4.40E−051.72E−04 2.18E−04 16.08 2.30E−04 7.50E−05 1.49E−04 7.20E−05 1.39E−041.33E−04 1.48E−04 3.80E−05 1.71E−04 2.07E−04 16.10 2.33E−04 6.40E−051.44E−04 9.60E−05 1.44E−04 1.17E−04 1.33E−04 4.10E−05 1.81E−04 2.04E−0416.12 2.19E−04 7.50E−05 1.48E−04 8.50E−05 1.26E−04 1.25E−04 1.45E−042.10E−05 1.69E−04 2.07E−04 16.13 2.45E−04 6.40E−05 1.45E−04 7.90E−051.42E−04 1.23E−04 1.34E−04 3.70E−05 1.71E−04 2.13E−04 16.15 2.21E−046.70E−05 1.51E−04 6.10E−05 1.50E−04 1.22E−04 1.39E−04 2.60E−05 1.81E−042.17E−04 16.17 2.28E−04 6.90E−05 1.28E−04 8.70E−05 1.39E−04 1.07E−041.25E−04 3.30E−05 1.66E−04 2.08E−04 16.18 2.17E−04 9.50E−05 1.44E−048.40E−05 1.41E−04 1.20E−04 1.42E−04 3.60E−05 1.69E−04 2.03E−04 16.202.28E−04 7.30E−05 1.48E−04 9.20E−05 1.33E−04 1.22E−04 1.49E−04 2.80E−051.86E−04 2.12E−04 16.22 2.19E−04 6.50E−05 1.30E−04 7.40E−05 1.21E−041.11E−04 1.40E−04 3.30E−05 1.70E−04 2.33E−04 16.23 2.09E−04 6.60E−051.41E−04 7.60E−05 1.50E−04 1.23E−04 1.30E−04 2.30E−05 1.64E−04 2.14E−0416.25 1.99E−04 7.90E−05 1.42E−04 7.70E−05 1.16E−04 1.17E−04 1.48E−043.50E−05 1.66E−04 2.07E−04 16.27 2.18E−04 5.80E−05 1.43E−04 7.60E−051.29E−04 1.25E−04 1.34E−04 3.30E−05 1.64E−04 2.12E−04 16.28 2.07E−046.40E−05 1.28E−04 7.40E−05 1.33E−04 1.13E−04 1.46E−04 3.10E−05 1.65E−042.13E−04 16.30 2.14E−04 6.60E−05 1.43E−04 8.60E−05 1.54E−04 1.09E−041.30E−04 3.00E−05 1.78E−04 2.04E−04 16.32 2.03E−04 5.60E−05 1.39E−047.30E−05 1.41E−04 1.17E−04 1.43E−04 2.30E−05 1.55E−04 2.10E−04 16.332.14E−04 6.60E−05 1.41E−04 8.00E−05 1.13E−04 1.03E−04 1.35E−04 2.40E−051.53E−04 2.08E−04 16.35 2.22E−04 6.50E−05 1.38E−04 7.90E−05 1.17E−041.20E−04 1.24E−04 3.10E−05 1.54E−04 2.02E−04 16.37 2.05E−04 5.00E−051.50E−04 7.20E−05 1.24E−04 1.17E−04 1.22E−04 4.10E−05 1.54E−04 2.09E−0416.38 1.92E−04 6.40E−05 1.55E−04 8.20E−05 1.24E−04 1.03E−04 1.33E−042.70E−05 1.58E−04 2.09E−04 16.40 1.99E−04 6.00E−05 1.45E−04 6.60E−051.24E−04 1.28E−04 1.25E−04 1.50E−05 1.46E−04 2.13E−04 16.42 2.00E−046.00E−05 1.53E−04 5.30E−05 1.14E−04 1.06E−04 1.22E−04 1.50E−05 1.53E−042.11E−04 16.43 2.05E−04 5.50E−05 1.40E−04 5.50E−05 1.29E−04 9.10E−051.33E−04 2.00E−05 1.43E−04 2.10E−04 16.45 2.03E−04 4.40E−05 1.31E−046.50E−05 1.31E−04 1.04E−04 1.20E−04 2.50E−05 1.50E−04 2.15E−04 16.471.99E−04 5.00E−05 1.39E−04 6.10E−05 1.20E−04 1.00E−04 1.25E−04 3.00E−051.48E−04 2.27E−04 16.48 2.05E−04 4.80E−05 1.50E−04 7.10E−05 1.46E−049.90E−05 1.28E−04 3.00E−05 1.41E−04 2.08E−04 16.50 2.12E−04 5.10E−051.40E−04 5.50E−05 1.21E−04 1.07E−04 1.20E−04 1.80E−05 1.49E−04 2.15E−0416.52 1.88E−04 5.00E−05 1.38E−04 5.50E−05 1.15E−04 1.13E−04 1.36E−041.60E−05 1.41E−04 2.12E−04 16.53 1.92E−04 5.40E−05 1.49E−04 5.10E−051.15E−04 1.11E−04 1.31E−04 2.60E−05 1.50E−04 2.01E−04 16.55 1.99E−045.60E−05 1.35E−04 5.60E−05 1.31E−04 1.13E−04 1.23E−04 2.40E−05 1.44E−042.20E−04 16.57 1.88E−04 6.90E−05 1.24E−04 5.40E−05 1.19E−04 1.22E−041.15E−04 3.00E−05 2.57E−04 2.17E−04 16.58 1.95E−04 5.30E−05 1.48E−044.80E−05 1.25E−04 1.17E−04 1.38E−04 2.00E−05 1.54E−04 2.04E−04 16.602.19E−04 5.80E−05 1.38E−04 5.00E−05 1.11E−04 1.04E−04 1.17E−04 1.10E−051.52E−04 2.05E−04 16.62 1.93E−04 6.50E−05 1.44E−04 5.50E−05 1.22E−041.05E−04 1.29E−04 1.60E−05 1.42E−04 2.15E−04 16.63 1.81E−04 3.60E−051.38E−04 5.40E−05 1.34E−04 1.10E−04 1.60E−04 1.20E−05 1.48E−04 2.04E−0416.65 1.78E−04 4.60E−05 1.42E−04 5.80E−05 1.31E−04 1.12E−04 1.14E−043.40E−05 1.43E−04 2.07E−04 16.67 1.99E−04 4.40E−05 1.51E−04 7.30E−051.14E−04 1.06E−04 1.24E−04 2.10E−05 1.53E−04 2.00E−04 16.68 1.92E−045.00E−05 1.32E−04 6.50E−05 1.10E−04 8.40E−05 1.31E−04 1.30E−05 1.41E−042.03E−04 16.70 1.93E−04 4.10E−05 1.38E−04 6.10E−05 1.12E−04 9.60E−051.10E−04 2.00E−06 1.42E−04 1.99E−04 16.72 2.02E−04 5.80E−05 1.44E−046.40E−05 1.16E−04 1.07E−04 1.21E−04 1.20E−05 1.55E−04 1.97E−04 16.731.85E−04 5.50E−05 1.44E−04 5.50E−05 1.11E−04 1.01E−04 1.03E−04 1.70E−051.46E−04 2.13E−04 16.75 2.01E−04 2.50E−05 1.38E−04 5.60E−05 1.24E−041.11E−04 1.20E−04 1.40E−05 1.32E−04 2.08E−04 16.77 1.81E−04 5.20E−051.50E−04 4.40E−05 1.07E−04 1.16E−04 1.06E−04 0.00E+00 1.28E−04 1.99E−0416.78 1.95E−04 6.40E−05 1.46E−04 5.30E−05 1.13E−04 1.07E−04 1.10E−041.70E−05 1.36E−04 2.03E−04 16.80 1.94E−04 4.30E−05 1.41E−04 4.40E−051.14E−04 9.40E−05 1.11E−04 1.00E−05 1.51E−04 2.04E−04 16.82 1.94E−045.50E−05 1.38E−04 4.30E−05 1.17E−04 1.00E−04 1.05E−04 1.30E−05 1.35E−042.04E−04 16.83 1.75E−04 6.00E−05 1.28E−04 4.00E−05 1.09E−04 9.90E−051.06E−04 1.30E−05 1.56E−04 2.12E−04 16.85 2.01E−04 4.60E−05 1.55E−043.10E−05 1.09E−04 9.70E−05 1.09E−04 3.00E−06 1.41E−04 1.94E−04 16.871.82E−04 6.30E−05 1.43E−04 5.10E−05 1.26E−04 9.10E−05 1.14E−04 1.60E−051.30E−04 1.94E−04 16.88 2.04E−04 4.60E−05 1.33E−04 3.70E−05 1.06E−041.00E−04 1.11E−04 1.10E−05 1.44E−04 1.83E−04 16.90 1.85E−04 4.10E−051.46E−04 3.70E−05 1.12E−04 9.20E−05 9.90E−05 6.00E−06 1.42E−04 1.98E−0416.92 1.78E−04 3.80E−05 1.44E−04 4.20E−05 1.11E−04 1.09E−04 9.10E−053.00E−05 1.31E−04 1.90E−04 16.93 1.93E−04 4.60E−05 1.39E−04 4.00E−058.90E−05 1.03E−04 1.20E−04 2.80E−05 1.36E−04 1.91E−04 16.95 1.94E−043.80E−05 1.50E−04 4.60E−05 1.02E−04 8.70E−05 1.07E−04 1.50E−05 1.30E−041.98E−04 16.97 1.85E−04 4.60E−05 1.38E−04 4.10E−05 1.06E−04 9.70E−051.03E−04 2.50E−05 1.32E−04 1.84E−04 16.98 1.85E−04 3.10E−05 1.29E−042.80E−05 1.23E−04 9.10E−05 1.06E−04 8.00E−06 1.25E−04 1.99E−04 17.002.01E−04 3.40E−05 1.46E−04 3.50E−05 1.00E−04 7.70E−05 9.70E−05 2.00E−051.29E−04 1.88E−04 17.02 1.90E−04 4.80E−05 1.40E−04 3.60E−05 1.12E−049.60E−05 1.07E−04 1.70E−05 1.30E−04 1.91E−04 17.03 2.08E−04 4.70E−051.55E−04 3.80E−05 1.10E−04 9.40E−05 1.11E−04 2.00E−06 1.35E−04 1.83E−0417.05 1.83E−04 3.50E−05 1.33E−04 6.10E−05 8.50E−05 8.70E−05 1.10E−045.00E−06 1.29E−04 1.85E−04 17.07 1.76E−04 3.60E−05 1.33E−04 3.20E−051.03E−04 9.20E−05 9.70E−05 2.10E−05 1.25E−04 1.93E−04 17.08 1.86E−045.30E−05 1.39E−04 3.30E−05 1.20E−04 9.40E−05 1.04E−04 2.20E−05 1.25E−041.92E−04 17.10 1.85E−04 4.80E−05 1.48E−04 4.30E−05 1.02E−04 8.50E−058.30E−05 1.50E−05 1.49E−04 1.91E−04 17.12 1.84E−04 4.80E−05 1.43E−045.30E−05 1.15E−04 8.00E−05 8.90E−05 7.00E−06 1.32E−04 1.85E−04 17.131.80E−04 4.40E−05 1.44E−04 3.60E−05 1.01E−04 8.30E−05 7.40E−05 7.00E−061.21E−04 1.90E−04 17.15 1.95E−04 4.80E−05 1.38E−04 4.30E−05 1.02E−049.30E−05 9.90E−05 −7.00E−06 1.45E−04 1.89E−04 17.17 1.83E−04 4.40E−051.40E−04 3.60E−05 1.03E−04 9.20E−05 9.00E−05 1.00E−06 1.20E−04 1.93E−0417.18 1.95E−04 4.20E−05 1.23E−04 3.30E−05 9.20E−05 9.70E−05 1.19E−04−2.00E−06 1.26E−04 1.82E−04 17.20 1.91E−04 4.20E−05 1.40E−04 3.80E−051.19E−04 8.10E−05 1.01E−04 −1.00E−05 1.25E−04 1.92E−04 17.22 1.91E−045.60E−05 1.44E−04 4.40E−05 1.10E−04 1.01E−04 1.05E−04 5.00E−06 1.25E−041.76E−04 17.23 1.93E−04 3.20E−05 1.26E−04 4.00E−05 1.16E−04 8.90E−059.60E−05 4.00E−06 1.13E−04 1.97E−04 17.25 1.91E−04 4.50E−05 1.44E−045.50E−05 1.12E−04 9.40E−05 1.07E−04 5.00E−06 1.28E−04 1.83E−04 17.271.80E−04 4.80E−05 1.40E−04 4.20E−05 1.09E−04 8.90E−05 9.00E−05 −7.00E−061.45E−04 1.75E−04 17.28 1.84E−04 3.50E−05 1.41E−04 6.60E−05 1.23E−049.10E−05 1.05E−04 3.00E−06 1.28E−04 1.88E−04 17.30 2.03E−04 3.20E−051.48E−04 7.00E−05 1.12E−04 9.70E−05 7.70E−05 8.00E−06 1.24E−04 1.89E−0417.32 2.00E−04 3.20E−05 1.44E−04 6.40E−05 1.32E−04 9.50E−05 1.00E−041.00E−06 1.42E−04 1.80E−04 17.33 2.00E−04 4.30E−05 1.43E−04 9.00E−051.36E−04 9.20E−05 9.30E−05 2.00E−06 1.21E−04 1.98E−04 17.35 1.94E−043.30E−05 1.52E−04 1.04E−04 1.22E−04 1.07E−04 9.60E−05 6.00E−06 1.25E−041.80E−04 17.37 1.91E−04 3.10E−05 1.42E−04 9.10E−05 1.46E−04 1.17E−049.30E−05 8.00E−06 1.24E−04 1.90E−04 17.38 1.86E−04 3.00E−05 1.33E−041.36E−04 1.46E−04 1.11E−04 1.07E−04 1.30E−05 1.23E−04 1.82E−04 17.401.88E−04 3.70E−05 1.38E−04 1.30E−04 1.66E−04 1.11E−04 9.10E−05 3.00E−061.31E−04 1.86E−04 17.42 2.01E−04 3.30E−05 1.39E−04 1.33E−04 1.60E−041.05E−04 9.40E−05 1.50E−05 1.28E−04 1.93E−04 17.43 1.73E−04 4.10E−051.36E−04 1.41E−04 1.43E−04 1.16E−04 9.70E−05 1.00E−05 1.23E−04 1.93E−0417.45 1.88E−04 4.00E−05 1.26E−04 1.49E−04 1.45E−04 1.17E−04 9.90E−056.00E−06 1.15E−04 1.92E−04 17.47 1.81E−04 4.00E−05 1.35E−04 1.28E−041.19E−04 1.17E−04 9.10E−05 5.00E−06 1.03E−04 1.81E−04 17.48 1.74E−043.40E−05 1.39E−04 1.20E−04 1.28E−04 1.10E−04 9.30E−05 5.00E−06 1.15E−041.74E−04 17.50 1.78E−04 4.10E−05 1.32E−04 1.14E−04 1.14E−04 1.07E−041.00E−04 1.00E−06 1.14E−04 1.88E−04 17.52 1.85E−04 3.70E−05 1.36E−041.11E−04 1.19E−04 1.15E−04 9.70E−05 2.00E−06 1.23E−04 1.83E−04 17.531.83E−04 3.30E−05 1.40E−04 9.10E−05 1.15E−04 1.07E−04 9.60E−05 0.00E+001.13E−04 1.83E−04 17.55 1.88E−04 3.80E−05 1.29E−04 8.40E−05 1.17E−049.60E−05 8.40E−05 3.00E−06 1.17E−04 1.84E−04 17.57 1.80E−04 4.40E−051.39E−04 8.90E−05 9.20E−05 8.10E−05 8.20E−05 5.00E−06 1.19E−04 1.79E−0417.58 1.82E−04 3.50E−05 1.33E−04 7.40E−05 1.02E−04 9.20E−05 9.20E−05−7.00E−06 1.24E−04 1.90E−04 17.60 1.78E−04 3.50E−05 1.17E−04 7.20E−059.50E−05 7.90E−05 9.70E−05 1.00E−05 1.22E−04 1.76E−04 17.62 1.79E−043.50E−05 1.21E−04 5.40E−05 8.60E−05 8.10E−05 9.00E−05 1.40E−05 1.09E−041.84E−04 17.63 1.72E−04 4.70E−05 1.11E−04 5.60E−05 9.40E−05 7.60E−056.20E−05 1.00E−05 1.04E−04 1.92E−04 17.65 1.80E−04 3.50E−05 1.23E−047.40E−05 9.00E−05 8.50E−05 9.00E−05 −7.00E−06 1.29E−04 1.65E−04 17.671.88E−04 3.40E−05 1.21E−04 5.20E−05 9.90E−05 9.00E−05 8.90E−05 8.00E−061.16E−04 1.61E−04 17.68 1.75E−04 2.60E−05 1.32E−04 4.60E−05 9.10E−058.40E−05 7.50E−05 3.00E−06 1.01E−04 1.72E−04 17.70 1.76E−04 4.20E−051.19E−04 5.70E−05 8.10E−05 8.90E−05 8.60E−05 1.00E−06 1.04E−04 1.69E−0417.72 1.55E−04 3.30E−05 1.31E−04 4.30E−05 8.30E−05 8.20E−05 8.70E−056.00E−06 1.15E−04 1.71E−04 17.73 1.76E−04 4.00E−05 1.24E−04 4.00E−059.20E−05 8.50E−05 8.60E−05 1.20E−05 1.11E−04 1.75E−04 17.75 1.59E−044.60E−05 1.20E−04 4.00E−05 9.90E−05 8.00E−05 7.90E−05 −8.00E−06 1.05E−041.64E−04 17.77 1.69E−04 3.80E−05 1.17E−04 3.00E−05 9.20E−05 7.70E−059.10E−05 3.00E−06 1.09E−04 1.73E−04 17.78 1.71E−04 4.00E−05 1.15E−044.20E−05 8.50E−05 7.90E−05 7.40E−05 −4.00E−06 1.14E−04 1.71E−04 17.801.82E−04 4.70E−05 1.24E−04 4.20E−05 1.04E−04 9.20E−05 6.70E−05 1.50E−051.05E−04 1.73E−04 17.82 1.75E−04 3.30E−05 1.09E−04 4.20E−05 9.70E−058.10E−05 6.90E−05 −3.00E−06 1.16E−04 1.73E−04 17.83 1.53E−04 3.60E−051.21E−04 4.40E−05 7.20E−05 8.50E−05 7.70E−05 −1.00E−06 1.07E−04 1.78E−0417.85 1.66E−04 3.80E−05 1.22E−04 3.60E−05 7.90E−05 9.20E−05 8.90E−055.00E−06 1.06E−04 1.73E−04 17.87 1.63E−04 3.40E−05 1.12E−04 5.30E−057.50E−05 7.90E−05 9.30E−05 −1.70E−05 1.16E−04 1.80E−04 17.88 1.56E−044.00E−05 1.11E−04 8.00E−05 7.30E−05 8.30E−05 7.20E−05 0.00E+00 1.16E−041.73E−04 17.90 1.60E−04 3.80E−05 1.10E−04 4.20E−05 7.10E−05 8.10E−057.40E−05 −6.00E−06 1.13E−04 1.86E−04 17.92 1.53E−04 2.30E−05 9.30E−054.30E−05 8.00E−05 9.10E−05 7.40E−05 0.00E+00 1.10E−04 1.66E−04 17.931.50E−04 3.40E−05 1.03E−04 3.60E−05 8.10E−05 8.00E−05 7.30E−05 −5.00E−061.07E−04 1.84E−04 17.95 1.51E−04 3.50E−05 1.07E−04 3.20E−05 6.60E−057.40E−05 7.40E−05 −4.00E−06 1.02E−04 1.71E−04 17.97 1.68E−04 5.50E−059.30E−05 5.30E−05 8.70E−05 9.90E−05 8.50E−05 −1.60E−05 1.17E−04 1.83E−0417.98 1.60E−04 2.50E−05 1.24E−04 3.80E−05 7.40E−05 7.70E−05 7.00E−05−7.00E−06 1.23E−04 1.59E−04 18.00 1.55E−04 3.60E−05 1.14E−04 4.30E−059.30E−05 8.20E−05 8.20E−05 −8.00E−06 1.05E−04 1.66E−04 18.02 1.51E−044.20E−05 1.09E−04 2.60E−05 7.20E−05 8.00E−05 6.60E−05 −3.00E−06 1.10E−041.76E−04 18.03 1.60E−04 1.80E−05 1.07E−04 2.70E−05 8.30E−05 7.90E−056.70E−05 1.10E−05 1.10E−04 1.74E−04 18.05 1.48E−04 4.40E−05 1.14E−043.60E−05 6.20E−05 6.90E−05 8.00E−05 −1.60E−05 9.70E−05 1.69E−04 18.071.55E−04 4.20E−05 1.09E−04 3.20E−05 7.20E−05 8.20E−05 6.70E−05 −4.00E−061.06E−04 1.66E−04 18.08 1.36E−04 3.80E−05 1.10E−04 3.20E−05 7.50E−058.10E−05 7.10E−05 1.10E−05 1.15E−04 1.81E−04 18.10 1.42E−04 1.70E−051.06E−04 4.00E−05 7.20E−05 6.30E−05 7.40E−05 −8.00E−06 1.17E−04 1.78E−0418.12 1.54E−04 3.60E−05 8.20E−05 4.30E−05 8.10E−05 9.50E−05 7.70E−05−1.30E−05 8.50E−05 1.85E−04 18.13 1.43E−04 3.40E−05 1.09E−04 4.80E−056.90E−05 6.70E−05 7.40E−05 −1.30E−05 1.03E−04 1.73E−04 18.15 1.55E−042.60E−05 1.03E−04 3.80E−05 6.40E−05 6.70E−05 7.40E−05 −5.00E−06 1.16E−041.56E−04 18.17 1.45E−04 4.00E−05 1.07E−04 4.20E−05 6.40E−05 7.40E−057.20E−05 −3.00E−06 1.11E−04 1.78E−04 18.18 1.53E−04 3.80E−05 1.00E−044.80E−05 7.60E−05 6.90E−05 6.40E−05 8.00E−06 8.50E−05 1.65E−04 18.201.62E−04 3.10E−05 1.10E−04 3.70E−05 7.00E−05 9.00E−05 6.30E−05 6.00E−061.07E−04 1.71E−04 18.22 1.54E−04 2.80E−05 9.40E−05 4.80E−05 8.20E−057.00E−05 6.90E−05 −3.00E−06 1.06E−04 1.71E−04 18.23 1.36E−04 2.50E−059.70E−05 4.00E−05 7.90E−05 6.60E−05 5.80E−05 −4.00E−06 1.00E−04 1.70E−0418.25 1.42E−04 3.00E−05 1.09E−04 3.30E−05 7.90E−05 9.40E−05 7.40E−05−5.00E−06 1.07E−04 1.83E−04 18.27 1.53E−04 3.30E−05 1.03E−04 2.30E−056.60E−05 7.00E−05 5.30E−05 1.50E−05 1.09E−04 1.82E−04 18.28 1.45E−041.60E−05 9.40E−05 4.40E−05 7.50E−05 6.40E−05 5.70E−05 −2.00E−06 1.13E−041.89E−04 18.30 1.52E−04 4.10E−05 8.30E−05 5.50E−05 7.40E−05 7.50E−057.20E−05 −1.00E−06 1.12E−04 1.73E−04 18.32 1.51E−04 2.50E−05 9.00E−054.60E−05 6.90E−05 9.00E−05 5.00E−05 −6.00E−06 1.23E−04 1.69E−04 18.331.44E−04 4.80E−05 9.90E−05 2.80E−05 6.10E−05 7.10E−05 5.60E−05 1.00E−051.02E−04 1.78E−04 18.35 1.42E−04 2.80E−05 1.03E−04 3.70E−05 7.20E−057.20E−05 6.60E−05 −6.00E−06 1.11E−04 1.64E−04 18.37 1.40E−04 2.60E−059.60E−05 3.20E−05 7.20E−05 7.00E−05 6.20E−05 −1.00E−05 1.02E−04 1.97E−0418.38 1.51E−04 3.40E−05 9.40E−05 3.50E−05 8.10E−05 7.20E−05 7.70E−05−1.60E−05 1.03E−04 1.80E−04 18.40 1.43E−04 3.20E−05 9.50E−05 3.50E−056.50E−05 7.00E−05 5.70E−05 −1.60E−05 1.16E−04 1.84E−04 18.42 1.48E−042.80E−05 9.10E−05 3.50E−05 6.40E−05 8.40E−05 6.30E−05 −3.00E−06 9.70E−051.74E−04 18.43 1.32E−04 3.40E−05 1.00E−04 3.40E−05 6.50E−05 8.40E−055.00E−05 −5.00E−06 1.10E−04 1.91E−04 18.45 1.53E−04 3.50E−05 1.03E−044.30E−05 6.40E−05 7.20E−05 6.20E−05 −1.50E−05 1.07E−04 1.76E−04 18.471.41E−04 3.10E−05 9.50E−05 4.20E−05 7.00E−05 8.10E−05 7.90E−05 −2.20E−051.04E−04 1.76E−04 18.48 1.26E−04 4.70E−05 9.00E−05 4.40E−05 6.10E−058.40E−05 7.70E−05 −7.00E−06 1.03E−04 1.79E−04 18.50 1.36E−04 4.20E−059.90E−05 1.01E−04 7.60E−05 7.50E−05 5.30E−05 7.00E−06 1.05E−04 1.83E−0418.52 1.38E−04 3.40E−05 1.01E−04 4.50E−05 6.90E−05 7.00E−05 5.70E−05−5.00E−06 9.90E−05 1.89E−04 18.53 1.26E−04 3.30E−05 9.90E−05 6.20E−057.00E−05 7.40E−05 6.40E−05 −2.10E−05 1.09E−04 1.61E−04 18.55 1.24E−044.60E−05 8.90E−05 4.10E−05 7.10E−05 5.60E−05 5.10E−05 −1.40E−05 1.04E−041.80E−04 18.57 1.36E−04 4.50E−05 9.40E−05 4.70E−05 6.50E−05 7.10E−055.80E−05 −3.00E−06 1.28E−04 1.84E−04 18.58 1.33E−04 4.00E−05 1.06E−045.40E−05 6.10E−05 7.50E−05 4.70E−05 −6.00E−06 1.11E−04 1.90E−04 18.601.29E−04 4.10E−05 1.01E−04 4.60E−05 7.50E−05 9.00E−05 7.60E−05 −6.00E−061.02E−04 1.80E−04 18.62 1.40E−04 4.20E−05 1.06E−04 3.70E−05 6.30E−057.10E−05 5.60E−05 −4.00E−06 1.03E−04 1.74E−04 18.63 1.34E−04 3.10E−059.60E−05 3.70E−05 5.80E−05 7.60E−05 5.00E−05 −2.00E−06 1.11E−04 1.90E−0418.65 1.26E−04 3.50E−05 8.90E−05 3.30E−05 5.70E−05 7.00E−05 4.30E−05−1.00E−05 1.03E−04 1.90E−04 18.67 1.40E−04 3.80E−05 9.00E−05 3.40E−055.00E−05 6.20E−05 5.10E−05 −4.00E−06 1.05E−04 1.83E−04 18.68 2.31E−044.30E−05 8.40E−05 4.80E−05 7.00E−05 7.40E−05 4.70E−05 −5.00E−06 9.60E−051.75E−04 18.70 1.24E−04 4.00E−05 9.40E−05 3.50E−05 7.10E−05 7.60E−056.30E−05 −1.30E−05 9.90E−05 1.83E−04 18.72 1.41E−04 4.60E−05 1.12E−044.10E−05 7.40E−05 6.50E−05 5.80E−05 −5.00E−06 1.10E−04 2.03E−04 18.731.56E−04 3.70E−05 9.40E−05 5.20E−05 5.60E−05 7.60E−05 6.40E−05 −3.00E−061.07E−04 1.66E−04 18.75 1.21E−04 2.60E−05 1.32E−04 3.80E−05 5.70E−055.50E−05 6.40E−05 −8.00E−06 1.00E−04 1.80E−04 18.77 1.13E−04 4.40E−058.90E−05 5.50E−05 7.10E−05 8.70E−05 5.10E−05 −1.00E−05 1.12E−04 1.83E−0418.78 1.33E−04 5.60E−05 1.03E−04 4.70E−05 6.00E−05 7.20E−05 4.30E−05−1.50E−05 9.60E−05 1.66E−04 18.80 1.38E−04 6.30E−05 8.30E−05 4.60E−057.50E−05 6.90E−05 5.20E−05 −1.50E−05 1.01E−04 1.86E−04 18.82 1.19E−046.50E−05 7.40E−05 6.10E−05 7.20E−05 5.30E−05 5.20E−05 −1.00E−05 1.05E−041.84E−04 18.83 1.34E−04 5.40E−05 8.70E−05 3.50E−05 6.20E−05 6.50E−055.80E−05 −2.10E−05 9.90E−05 1.81E−04 18.85 1.24E−04 5.00E−05 1.14E−044.10E−05 5.40E−05 5.30E−05 6.40E−05 −3.00E−06 9.60E−05 1.83E−04 18.871.21E−04 5.50E−05 9.50E−05 2.80E−05 6.60E−05 5.30E−05 5.30E−05 −1.70E−059.70E−05 1.70E−04 18.88 1.38E−04 5.40E−05 9.60E−05 4.40E−05 5.00E−057.70E−05 6.00E−05 −6.00E−06 1.07E−04 1.76E−04 18.90 1.33E−04 4.50E−058.60E−05 4.50E−05 5.80E−05 8.00E−05 5.30E−05 7.00E−06 9.20E−05 1.85E−0418.92 1.43E−04 6.10E−05 8.20E−05 3.10E−05 5.10E−05 6.90E−05 4.30E−05−8.00E−06 1.07E−04 1.83E−04 18.93 1.22E−04 5.60E−05 8.50E−05 4.10E−055.10E−05 5.60E−05 5.40E−05 −1.10E−05 9.50E−05 1.78E−04 18.95 1.16E−047.40E−05 9.70E−05 5.80E−05 7.00E−05 7.30E−05 7.90E−05 −1.70E−05 9.70E−051.76E−04 18.97 1.35E−04 4.70E−05 7.70E−05 4.00E−05 5.80E−05 8.10E−055.80E−05 −1.30E−05 1.09E−04 1.92E−04 18.98 1.28E−04 6.30E−05 8.50E−055.10E−05 6.20E−05 7.90E−05 6.70E−05 −1.20E−05 1.07E−04 1.73E−04 19.001.14E−04 6.50E−05 8.60E−05 3.20E−05 8.00E−05 6.90E−05 4.80E−05 −2.10E−051.17E−04 1.72E−04 19.02 1.21E−04 6.60E−05 8.70E−05 4.50E−05 5.60E−056.40E−05 5.20E−05 −2.70E−05 1.05E−04 1.79E−04 19.03 1.05E−04 7.10E−057.90E−05 6.30E−05 4.70E−05 8.10E−05 6.10E−05 −1.00E−05 1.06E−04 1.85E−0419.05 1.14E−04 5.80E−05 8.00E−05 6.10E−05 5.10E−05 7.00E−05 6.70E−05−1.30E−05 1.14E−04 1.72E−04 19.07 1.33E−04 7.30E−05 7.40E−05 4.10E−056.10E−05 6.10E−05 5.80E−05 −1.60E−05 1.01E−04 1.86E−04 19.08 1.28E−045.80E−05 9.30E−05 3.80E−05 6.40E−05 7.00E−05 5.60E−05 −2.70E−05 1.02E−041.81E−04 19.10 1.26E−04 7.00E−05 8.10E−05 4.40E−05 6.00E−05 6.70E−055.40E−05 −7.00E−06 1.03E−04 1.70E−04 19.12 1.24E−04 5.70E−05 9.70E−053.10E−05 6.20E−05 7.70E−05 6.00E−05 −3.20E−05 1.03E−04 1.82E−04 19.131.15E−04 6.60E−05 7.00E−05 5.10E−05 8.40E−05 6.10E−05 5.70E−05 −2.20E−051.17E−04 1.98E−04 19.15 1.16E−04 6.60E−05 8.10E−05 4.10E−05 5.20E−055.70E−05 5.00E−05 −2.60E−05 1.15E−04 1.83E−04 19.17 1.21E−04 7.20E−058.30E−05 4.40E−05 5.80E−05 6.30E−05 4.30E−05 −1.50E−05 1.05E−04 1.79E−0419.18 1.15E−04 6.70E−05 8.60E−05 5.10E−05 4.20E−05 7.90E−05 6.10E−05−6.00E−06 1.07E−04 1.66E−04 19.20 1.34E−04 6.70E−05 8.60E−05 4.80E−056.40E−05 5.60E−05 5.70E−05 −1.50E−05 9.90E−05 1.73E−04 19.22 1.14E−046.70E−05 8.50E−05 4.20E−05 6.50E−05 6.30E−05 5.20E−05 −2.60E−05 1.05E−041.89E−04 19.23 1.29E−04 7.50E−05 8.50E−05 4.20E−05 5.20E−05 7.20E−055.30E−05 −1.60E−05 9.30E−05 1.78E−04 19.25 1.14E−04 7.90E−05 8.00E−053.80E−05 4.60E−05 7.50E−05 6.70E−05 −2.80E−05 9.30E−05 1.76E−04 19.271.15E−04 6.90E−05 8.20E−05 4.70E−05 5.60E−05 5.70E−05 5.70E−05 −2.10E−051.00E−04 1.89E−04 19.28 1.13E−04 7.60E−05 8.10E−05 4.60E−05 5.00E−058.10E−05 5.60E−05 −2.30E−05 1.02E−04 1.61E−04 19.30 1.26E−04 8.40E−058.30E−05 3.20E−05 5.50E−05 5.70E−05 5.40E−05 −1.70E−05 1.10E−04 1.74E−0419.32 1.09E−04 5.50E−05 8.60E−05 3.00E−05 5.60E−05 7.20E−05 6.40E−05−2.20E−05 1.10E−04 1.71E−04 19.33 1.15E−04 4.80E−05 7.60E−05 3.50E−056.10E−05 5.30E−05 4.80E−05 −3.20E−05 1.15E−04 1.58E−04 19.35 1.16E−046.60E−05 8.90E−05 4.10E−05 5.30E−05 6.50E−05 5.10E−05 −2.10E−05 1.03E−041.79E−04 19.37 1.23E−04 5.50E−05 8.70E−05 5.00E−05 4.40E−05 5.60E−056.40E−05 1.40E−05 9.90E−05 1.73E−04 19.38 1.44E−04 7.50E−05 8.00E−055.40E−05 4.80E−05 6.00E−05 4.30E−05 −1.20E−05 9.30E−05 1.71E−04 19.401.23E−04 5.60E−05 9.60E−05 3.10E−05 5.50E−05 6.30E−05 6.10E−05 −1.10E−051.05E−04 1.75E−04 19.42 1.24E−04 8.10E−05 6.50E−05 3.60E−05 4.00E−056.30E−05 4.50E−05 −1.50E−05 1.02E−04 1.71E−04 19.43 1.09E−04 6.30E−057.20E−05 4.30E−05 4.80E−05 7.20E−05 5.20E−05 −1.40E−05 9.30E−05 1.84E−0419.45 1.19E−04 7.90E−05 8.00E−05 5.40E−05 5.80E−05 5.80E−05 5.50E−05−1.30E−05 1.05E−04 1.79E−04 19.47 1.09E−04 7.10E−05 7.40E−05 3.80E−057.10E−05 7.20E−05 5.60E−05 −7.00E−06 1.01E−04 1.84E−04 19.48 1.23E−045.80E−05 7.00E−05 3.20E−05 5.10E−05 5.50E−05 5.70E−05 −1.50E−05 1.00E−041.60E−04 19.50 1.15E−04 7.30E−05 7.40E−05 5.70E−05 6.60E−05 6.30E−054.50E−05 −2.20E−05 1.01E−04 1.68E−04 19.52 1.21E−04 6.10E−05 8.00E−053.50E−05 6.30E−05 6.20E−05 6.50E−05 −2.70E−05 9.70E−05 1.84E−04 19.531.25E−04 7.30E−05 7.40E−05 4.50E−05 7.60E−05 7.60E−05 7.60E−05 −1.50E−059.90E−05 1.78E−04 19.55 1.07E−04 7.90E−05 9.10E−05 4.10E−05 7.10E−055.50E−05 4.40E−05 −1.40E−05 9.30E−05 1.71E−04 19.57 1.19E−04 7.50E−058.70E−05 5.50E−05 5.80E−05 7.00E−05 6.40E−05 −1.70E−05 9.50E−05 1.62E−0419.58 1.06E−04 7.60E−05 7.50E−05 3.70E−05 6.40E−05 6.70E−05 6.70E−05−1.70E−05 1.04E−04 1.71E−04 19.60 1.23E−04 6.90E−05 7.90E−05 4.30E−056.60E−05 7.70E−05 6.50E−05 −1.20E−05 9.50E−05 1.69E−04 19.62 1.12E−048.10E−05 8.30E−05 3.20E−05 6.60E−05 6.70E−05 6.40E−05 −1.70E−05 1.04E−041.82E−04 19.63 9.30E−05 9.70E−05 7.90E−05 3.80E−05 5.60E−05 8.30E−055.80E−05 −1.80E−05 1.00E−04 2.29E−04 19.65 1.09E−04 7.10E−05 6.10E−053.40E−05 5.40E−05 6.60E−05 6.30E−05 −1.30E−05 9.30E−05 1.73E−04 19.671.15E−04 7.30E−05 8.10E−05 4.50E−05 7.20E−05 7.90E−05 5.30E−05 −1.00E−051.01E−04 1.78E−04 19.68 1.14E−04 7.60E−05 7.20E−05 3.80E−05 5.40E−058.50E−05 6.20E−05 −1.80E−05 9.10E−05 1.78E−04 19.70 9.90E−05 7.90E−059.10E−05 4.40E−05 6.70E−05 6.30E−05 6.50E−05 −7.00E−06 9.20E−05 1.75E−0419.72 1.14E−04 7.60E−05 6.40E−05 4.20E−05 6.50E−05 7.20E−05 5.70E−05−1.80E−05 9.40E−05 1.65E−04 19.73 1.00E−04 7.20E−05 8.50E−05 5.20E−058.00E−05 6.70E−05 5.30E−05 −1.30E−05 9.00E−05 1.75E−04 19.75 1.02E−047.90E−05 7.70E−05 2.70E−05 7.00E−05 8.00E−05 6.20E−05 −2.50E−05 1.05E−041.76E−04 19.77 9.70E−05 8.60E−05 5.60E−05 4.10E−05 7.10E−05 9.10E−055.20E−05 −1.30E−05 1.06E−04 1.71E−04 19.78 1.15E−04 8.50E−05 7.50E−053.20E−05 8.10E−05 7.20E−05 5.30E−05 −8.00E−06 9.90E−05 1.63E−04 19.801.13E−04 7.60E−05 8.20E−05 3.20E−05 9.20E−05 9.00E−05 5.60E−05 −1.70E−058.70E−05 1.69E−04 19.82 1.16E−04 7.20E−05 7.00E−05 3.50E−05 7.70E−058.20E−05 5.00E−05 −2.30E−05 1.01E−04 1.73E−04 19.83 1.23E−04 6.50E−056.10E−05 3.00E−05 7.60E−05 8.30E−05 4.70E−05 −1.60E−05 9.30E−05 1.59E−0419.85 1.26E−04 7.70E−05 7.10E−05 3.00E−05 8.00E−05 7.70E−05 4.70E−05−1.80E−05 1.02E−04 1.73E−04 19.87 1.23E−04 6.60E−05 8.00E−05 4.60E−057.50E−05 7.70E−05 5.80E−05 −8.00E−06 1.00E−04 1.74E−04 19.88 1.24E−047.40E−05 7.10E−05 1.20E−05 7.60E−05 7.60E−05 6.00E−05 −1.30E−05 9.10E−051.74E−04 19.90 1.15E−04 6.10E−05 6.60E−05 3.20E−05 7.70E−05 7.40E−055.40E−05 −2.30E−05 8.70E−05 2.01E−04 19.92 1.34E−04 6.30E−05 6.90E−053.80E−05 8.40E−05 7.40E−05 5.80E−05 −2.30E−05 1.03E−04 1.62E−04 19.931.26E−04 8.70E−05 8.30E−05 3.00E−05 8.20E−05 8.60E−05 5.10E−05 −1.50E−051.04E−04 1.55E−04 19.95 1.01E−04 6.50E−05 6.20E−05 2.10E−05 7.30E−059.20E−05 5.70E−05 −1.10E−05 1.04E−04 1.78E−04 19.97 1.10E−04 8.10E−058.10E−05 3.20E−05 7.10E−05 8.50E−05 5.00E−05 −1.10E−05 9.00E−05 1.61E−0419.98 1.25E−04 8.10E−05 6.20E−05 2.70E−05 7.70E−05 6.90E−05 4.40E−05−1.80E−05 1.02E−04 1.56E−04 20.00 1.06E−04 6.20E−05 7.10E−05 2.80E−059.10E−05 6.70E−05 5.60E−05 −1.50E−05 1.05E−04 1.71E−04

The advantage of SEC-HPLC analysis was that the insufficiently PEGylatedbatches, e.g. containing less than 5 PEGylated sites per htCBS C15Sprotein and/or containing any unmodified htCBS C15S, were characterizedby longer retention time than the desired ones and were swiftlyidentified for rescue by adding more PEG molecules to the reactionmixture (in-process monitoring of PEGylation). The unmodified htCBS C15Ssample was observed to have a retention time of 11.97 minutes. Thesample from the 6-70 and 8-23 batches were observed to have a retentiontime of 9.67 minutes. The sample from the 6-89 batch was observed tohave a retention time of 9.88 minutes. The sample from the 8-22 batchwas observed to have a retention time of 9.95 minutes. Therefore, aretention time of greater than 9.67 minutes was determined to indicateinsufficient PEGylation.

The sample from the 8-14 batch was observed to have a retention time of9.57 minutes. The sample from the 8-15 batch was observed to have aretention time of 9.53 minutes. The sample from the 8-16 batch wasobserved to have a retention time of 9.55 minutes. The sample from the8-24 batch was observed to have a retention time of 9.55 minutes. Thesample from the 8-25 batch was observed to have a retention time of 9.58minutes. Therefore, a retention time of less than 9.53 minutes wasdetermined to indicate acceptable PEGylation.

Another method was based on non-reduced capillary electrophoresisallowing resolution and quantification of multiple 20NHS PEG-htCBS C15Sspecies (FIG. 1 ). Insufficient PEGylation of the batch wascharacterized by the presence of unmodified enzyme and an increasedoccurrence of low molecular weight 20NHS PEG-htCBS C15S species.Therefore, this method was found suitable to define strict acceptancecriteria for the 20NHS PEG-htCBS C15S final product.

The protein sequence of htCBS C15S contains 30 lysine residues, whichall represent potential sites for NHS ester PEGylation. To identify thelysine residues involved in PEGylation, LC-UV-MS was performed three20NHS PEG-htCBS C15S batches. After de-PEGylation, the formerlyPEGylated lysines were left with a linker that enabled theirdifferentiation from unmodified lysines and quantification via peptidemapping after cleavage with Asp-N endopeptidase and comparison tosimilarly processed unmodified htCBS C15S. LC-MS analysis was performedafter Asp-N digest of unmodified htCBS C15S as a reference protein(control).

Table 2 shows the relative abundance of peptides and was used tocalculate the number of PEGs per peptide for the reference enzyme, analkylated, unmodified htCBS C15S, identified by LC/MS/MS. (RT=retentiontime).

TABLE 2 NHS ester PEGylation mapping of a reference enzyme SequenceSEQ ID Theoretical Observed Δ mass RT Location Sequence NO Mass (Da)Mass (Da) (ppm) (min) Abundance  2-34 PSETPQAEVGPTGSPHRSGPHSAKGSLE 23294.59 3294.59 0.64 33.8 462828 KGSPE  9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE3 2584.26 2584.26 0.07 30.8 449108 35-46 DKEAKEPLWIRP 4 1480.80 1480.80−0.08 50.6 323088 40-46 EPLWIRP 5 909.51 909.51 0.71 54.5 1369777 47-78DAPSRCTWQLGRPASESPHHHTAPAKS 6 3528.78 3528.78 0.13 49 1805933 PKILP79-85 DILKKIG 7 785.50 785.50 0.06 41.9 1034520  86-106DTPMVRINKIGKKFGLKCELL 8 2459.38 2459.38 0.07 59.8 752660 107-119AKCEFFNAGGSVK 9 1413.67 1413.67 0.28 41.5 865418 120-128 DRISLRMIE 101131.61 1131.61 0.74 51.3 1259968 120-128 DRISLRMIE 10 1147.60 1147.600.34 43.8 42144 133-139 DGTLKPG 11 686.36 686.36 0.72 24 665344 140-178DTIIEPTSGNTGIGLALAAAVRGYRCIIV 12 4147.15 4147.15 −0.29 92.2 422022MPEKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 13 2098.16 2098.17 0.21 64.23651887 179-197 DVLRALGAEIVRTPTNARF 13 2099.15 2099.15 1.17 63.7 1401992198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2597.34 2597.33 −0.2 58.1 1160943198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2598.32 2598.32 0.17 58.8 543933221-233 DQYRNASNPLAHY 15 1547.71 1547.71 0.22 40.9 209797 221-233DQYRNASNPLAHY 15 1548.70 1548.70 1.59 40.3 156242 221-233 DQYRNASNPLAHY15 1548.70 1548.70 0.79 41.2 708853 238-244 DEILQQC 16 904.40 904.400.12 33.1 258834 245-248 DGKL 17 431.24 431.24 0.01 19.7 98742 249-269DMLVASVGTGGTITGIARKLK 18 2087.18 2087.18 0.04 59.3 458054 249-269DMLVASVGTGGTITGIARKLK 18 2103.17 2103.17 0.42 55.3 112910 270-301EKCPGCRIIGVDPEGSILAEPEELNQTEQ 19 3632.70 3632.70 −0.18 57.7 99941 TTY302-308 EVEGIGY 20 765.35 765.36 0.99 38.3 356953 309-315 DFIPTVL 21803.44 803.44 −0.02 66.5 1980222 316-320 DRTW 22 588.32 588.32 0.75 27.3891244 321-327 DKWFKSN 23 923.45 923.45 0.03 35.3 209620 328-365DEEAFTFARMLIAQEGLLCGGSAGSTV 24 3937.96 3937.95 −0.26 95.1 200461AVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.88 1735.87 −1.79 47.2 212890363-375 ELQEGQRCVVILP 26 1539.81 1539.81 0.31 55.7 81479 366-375EGQRCVVILP 27 1169.62 1169.62 −0.36 53.8 375007 376-387 DSVRNYMTKFLS 281459.71 1459.71 −0.15 55.3 1194612 388-400 DRWMLQKGFLKEE 29 1678.851678.85 0.44 52.2 836829 388-400 DRWMLQKGFLKEE 29 1694.85 1694.85 0.3949.4 129978 401-413 DLTEKKPWWWHLR 30 1793.94 1793.94 0.19 56.7 2238451

Theoretical molecular masses (mono-isotopic) were calculated usingMassHunter BioConfirm software (Agilent, Santa Clara, Calif.). Theobserved masses (mono-isotopic) were within 5 ppm of their theoreticalvalue. Abundances were determined using MassHunter software (Agilent,Santa Clara, Calif.). Peptide 120-128 was observed to have themodification of a 1*oxidation. Peptide 179-197 was observed to have themodification of a 1*deamidation. Peptide 198-220 was observed to havethe modification of a 1*deamidation. Peptide 221-233 was observed tohave the modification of a 1*deamidation. Peptide 221-233 was observedto have the modification of a 1*deamidation 249-269 was observed to havethe modification of a 1*oxidation. Peptide 388-400 was observed to havethe modification of a 1*oxidation.

LC-MS analysis was performed after Asp-N digest of reduced, alkylated,PEGylated 20NHS PEG-htCBS C15S (batch #1). Table 3 shows the relativeabundance of peptides and was used to calculate the number of PEGs perpeptide for an Asp-N digest of reduced, alkylated, PEGylated 20NHSPEG-htCBS C15S (batch #1), identified by LC/MS/MS.

TABLE 3NHS ester PEGylation mapping of Asp-N digest of reduced, alkylated, PEGylated20NHS PEG-htCBS C15S (batch #1) Sequence SEQ Theoretical Observed Δ massRT Location Sequence ID NO Mass (Da) Mass (Da) (PPm) (min) Abundance 2-34 PSETPQAEVGPTGSPHRSGPHSAKGS 2 3294.59 3294.59 −0.46 33.8 74988LEKGSPE  2-34 PSETPQAEVGPTGSPHRSGPHSAKGS 2 3408.62 3408.62 1.2 36.7285758 LEKGSPE  2-34 PSETPQAEVGPTGSPHRSGPHSAKGS 2 3522.65 3522.65 0.5139.3 251006 LEKGSPE  9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE 3 2584.26 2584.260.43 30.8 50722  9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE 3 2698.29 2698.3 1.3434.6 156217  9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE 3 2812.33 2812.33 0.43 37.659784 35-46 DKEAKEPLWIRP 4 1480.8 1480.8 0.14 50.5 419366 35-46DKEAKEPLWIRP 4 1594.84 1594.83 −0.42 53.3 115586 40-46 EPLWIRP 5 909.51909.51 0.36 54.4 1267685 47-78 DAPSRCTWQLGRPASESPHHHTAPA 6 3528.783528.78 0.35 49 916229 KSPKILP 47-78 DAPSRCTWQLGRPASESPHHHTAPA 6 3642.813642.81 0.3 51.2 536395 KSPKILP 79-85 DILKKIG 7 785.5 785.5 0.2 41.91447731 79-85 DILKKIG 7 899.53 899.53 0.01 47.1 49654  86-106DTPMVRINKIGKKFGLKCELL 8 2459.38 2459.38 0 59.8 278431  86-106DTPMVRINKIGKKFGLKCELL 8 2573.41 2573.41 0.2 63.4 207777  86-106DTPMVRINKIGKKFGLKCELL 8 2573.41 2573.41 0.39 66 268441 107-119AKCEFFNAGGSVK 9 1413.67 1413.67 0.21 41.5 1031048 120-128 DRISLRMIE 101131.61 1131.61 0.63 51.2 1738225 120-128 DRISLRMIE 10 1147.6 1147.6−0.19 43.8 74835 133-139 DGTLKPG 11 686.36 686.36 0.53 24 837583 140-178DTIIEPTSGNTGIGLALAAAVRGYRCI 12 4147.15 4147.15 −0.03 92.2 575055IVMPKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 13 2098.16 2098.17 0.18 64.14896736 179-197 DVLRALGAEIVRTPTNARF 13 2099.15 2099.15 0.59 63.6 1870150198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2597.34 2597.34 0.11 58 1302818198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2598.32 2598.32 0.17 58.7 591137198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2711.37 2711.37 1 63.5 65301 221-233DQYRNASNPLAHY 15 1547.71 1547.71 −0.06 40.9 256919 221-233 DQYRNASNPLAHY15 1548.7 1548.7 1.11 40.3 198652 221-233 DQYRNASNPLAHY 15 1548.7 1548.70.61 41.2 802841 238-244 DEILQQC 16 904.4 904.4 −0.35 33.1 216368245-248 DGKL 17 431.24 431.24 0.15 19.9 75839 245-248 DGKL 17 545.27545.27 −0.29 31.7 73171 249-269 DMLVASVGTGGTITGIARKLK 18 2087.18 2087.18−0.18 59.2 458170 249-269 DMLVASVGTGGTITGIARKLK 18 2103.17 2103.17 0.1655.2 130431 270-301 EKCPGCRIIGVDPEGSILAEPEELNQT 19 3632.7 3632.7 1.2457.6 37257 EQTTY 270-301 EKCPGCRIIGVDPEGSILAEPEELNQT 19 3746.73 3746.72−1.28 59.5 23764 EQTTY 302-308 EVEGIGY 20 765.35 765.35 0.03 38.3 274612309-315 DFIPTVL 21 803.44 803.44 0.29 66.4 2313422 316-320 DRTW 22588.32 588.32 0.11 27.2 1216696 321-327 DKWFKSN 23 923.45 923.45 0.0335.3 100188 321-327 DKWFKSN 23 1037.48 1037.48 −0.64 41.9 44718 328-365DEEAFTFARMLIAQEGLLCGGSAGS 24 3937.96 3937.95 −0.4 95.1 82719TVAVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.88 1735.87 −1.84 47.2 158013363-375 ELQEGQRCVVILP 26 1539.81 1539.81 0.05 55.6 109572 366-375EGQRCVVILP 27 1169.62 1169.62 0.17 53.7 425023 376-387 DSVRNYMTKFLS 281459.71 1459.71 0.34 55.2 1488072 388-400 DRWMLQKGFLKEE 29 1678.851678.85 −0.04 52.1 1018180 388-400 DRWMLQKGFLKEE 29 1694.85 1694.85−0.05 49.2 138406 388-400 DRWMLQKGFLKEE 29 1792.88 1792.88 −0.32 57.461595 401-413 DLTEKKPWWWHLR 30 1793.94 1793.94 0.13 56.7 971171 401-413DLTEKKPWWWHLR 30 1907.97 1907.97 0.08 60.8 1558334 401-413 DLTEKKPWWWHLR30 2022 2022 0.4 66.5 242737

Further, 5 peptides with 2 lysines and 1 peptide with 4 lysines wereidentified, of which one lysine residue was found inconsistentlyPEGylated: K36/39, K72/75, K82/83, K94/97/98/102, K322/325 and K394/398.The estimated number of PEG/peptide was calculated using the followingformula: Estimated (PEG/peptide)ratio=(Abundance(1*Linker)+2*Abundance(2*Linker))/((Abundance(NoLinker)+Abundance(1*Linker)+Abundance(2*Linker)). Since PEGs are knownin the art to not migrate according to their molecular weight in gels orchromatographic applications due to a significant solvation envelope andthus increased hydrodynamic radius, the extent of PEGylation wasestimated to be in the range of 5.0±0.5 PEGs per CBS monomer.Specifically, the number PEG molecules per peptide was estimated to be1.29 for 2-34, 1.30 for 9-34, 0.22 for 35-46, 0.37 for 47-78, 0.03 for79-85 and 198-220, 0.63 for 86-106, 0.49 for 245-248, 0.39 for 270-301,0.31 for 321-327, and 0.05 for 388-400.

Peptide 2-34 was observed to have the modification of a 1*linker.Peptide 2-34 was observed to have the modification of a 2*linker.Peptide 9-34 was observed to have the modification of a 1*linker.Peptide 9-34 was observed to have the modification of a 2*linker.Peptide 35-46 was observed to have the modification of a 1*linker.Peptide 47-78 was observed to have the modification of a 1*linker.Peptide 79-85 was observed to have the modification of a 1*linker.Peptide 86-106 was observed to have the modification of a 1*linker.Peptide 86-106 was observed to have the modification of a 1*linker.Peptide 120-128 was observed to have the modification of a 1*oxidation.Peptide 179-197 was observed to have the modification of a1*deamidation. Peptide 198-220 was observed to have the modification ofa 1*deamidation. Peptide 198-220 was observed to have the modificationof a 1*linker. Peptide 221-233 was observed to have the modification ofa 1*deamidation. Peptide 221-233 was observed to have the modificationof a 1*deamidation. Peptide 245-248 was observed to have themodification of a 1*linker. Peptide 249-269 was observed to have themodification of a 1*oxidation. Peptide 270-301 was observed to have themodification of a 1*linker. Peptide 321-327 was observed to have themodification of a 1*linker. Peptide 388-400 was observed to have themodification of a 1*oxidation. Peptide 388-400 was observed to have themodification of a 1*linker. Peptide 401-413 was observed to have themodification of a 1*linker. Peptide 401-413 was observed to have themodification of a 2*linker.

LC-MS analysis was performed after Asp-N digest of reduced, alkylated,PEGylated 20NHS PEG-htCBS C15S (batch #2). Table 4 shows the relativeabundance of peptides and estimated number of PEGs per peptide for anAsp-N digest of a reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S(batch #2), identified by LC/MS/MS.

TABLE 4NHS ester PEGylation mapping of Asp-N digest of reduced, alkylated, PEGylated20NHS PEG-htCBS C15S (batch #2) Sequence SEQ ID Theoretical ObservedΔ mass RT Location Sequence NO. Mass (Da) Mass (Da) (ppm) (min)Abundance  2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL 2 3294.5865 3294.5858 −0.2134.1 91654 EKGSPE  2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL 2 3408.61823408.6173 −0.26 37.1 212552 EKGSPE  2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL 23522.6499 3522.6505 0.17 39.6 116731 EKGSPE  9-34EVGPTGSPHRSGPHSAKGSLEKGSPE 3 2584.263 2584.2621 −0.35 31.2 110005  9-34EVGPTGSPHRSGPHSAKGSLEKGSPE 3 2698.2947 2698.2944 −0.1 35 207053  9-34EVGPTGSPHRSGPHSAKGSLEKGSPE 3 2812.3264 2812.3283 0.68 37.6 50468 35-46DKEAKEPLWIRP 4 1480.8038 1480.804 0.15 51.1 130726 35-46 DKEAKEPLWIRP 41594.8355 1594.836 0.31 53.8 26208 40-46 EPLWIRP 5 909.5072 909.50750.31 54.9 370392 47-78 DAPSRCTWQLGRPASESPHHHTAPAK 6 3528.7797 3528.7769−0.79 49.4 608763 SPKILP 47-78 DAPSRCTWQLGRPASESPHHHTAPAK 6 3642.81143642.8084 −0.82 51.6 211918 SPKILP 79-85 DILKKIG 7 785.5011 785.50170.78 42.3 454812 79-85 DILKKIG 7 899.5328 899.5338 1.11 47.5 11289 86-106 DTPMVRINKIGKKFGLKCELL 8 2459.3756 2459.3774 0.73 60.8 179277 86-106 DTPMVRINKIGKKFGLKCELL 8 2573.4073 2573.4077 0.16 64.6 92660 86-106 DTPMVRINKIGKKFGLKCELL 8 2573.4073 2573.4068 −0.19 67.3 117060107-119 AKCEFFNAGGSVK 9 1413.6711 1413.6716 0.35 42 495351 120-128DRISLRMIE 10 1131.607 1131.6075 0.46 51.7 664834 120-128 DRISLRMIE 101147.6019 1147.6023 0.35 44.3 33046 133-139 DGTLKPG 11 686.3599 686.36030.61 24.3 285635 140-178 DTIIEPTSGNTGIGLALAAAVRGYRCII 12 4147.1484147.1451 −0.7 93.5 181549 VMPEKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 132098.1647 2098.1657 0.49 65.3 2248724 179-197 DVLRALGAEIVRTPTNARF 132099.1487 2099.1508 0.99 64.8 936714 198-220 DSPESHVGVAWRLKNEIPNSHIL 142597.335 2597.3345 −0.23 58.7 635630 198-220 DSPESHVGVAWRLKNEIPNSHIL 142598.3191 2598.3202 0.45 59.5 352959 198-220 DSPESHVGVAWRLKNEIPNSHIL 142711.3667 2711.3649 −0.66 64.5 23552 221-233 DQYRNASNPLAHY 15 1547.71171547.7118 0.06 41.4 128480 221-233 DQYRNASNPLAHY 15 1548.6957 1548.69710.9 40.8 111326 221-233 DQYRNASNPLAHY 15 1548.6957 1548.6955 −0.17 41.7405793 238-244 DEILQQC 16 904.396 904.3962 0.23 33.5 126894 245-248 DGKL17 431.238 431.2381 0.12 20.5 40770 245-248 DGKL 17 545.2697 545.26990.37 32.2 26289 249-269 DMLVASVGTGGTITGIARKLK 18 2087.1773 2087.178 0.3459.8 301153 249-269 DMLVASVGTGGTITGIARKLK 18 2103.1722 2103.174 0.8655.3 81381 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3632.6975 3632.69960.58 58.1 51146 QTTY 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3746.72923746.7269 −0.61 60 18541 QTTY 302-308 EVEGIGY 20 765.3545 765.3549 0.4938.7 244178 309-315 DFIPTVL 21 803.4429 803.4436 0.86 66.9 1102772316-320 DRTW 22 588.3231 588.3235 0.59 27.7 482942 321-327 DKWFKSN 23923.4501 923.4504 0.33 35.9 41312 321-327 DKWFKSN 23 1037.4818 1037.48260.77 42.5 10692 328-365 DEEAFTFARMLIAQEGLLCGGSAGST 24 3937.95543937.9519 −0.89 95.6 35286 VAVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.8751735.8712 −2.19 47.7 113391 363-375 ELQEGQRCVVILP 26 1539.8079 1539.80830.26 55.9 44354 366-375 EGQRCVVILP 27 1169.6227 1169.6238 0.95 54.1143135 376-387 DSVRNYMTKFLS 28 1459.713 1459.7144 0.96 56.1 679328388-400 DRWMLQKGFLKEE 29 1678.8501 1678.8505 0.22 52.8 428907 388-400DRWMLQKGFLKEE 29 1694.845 1694.8454 0.24 69.9 80607 388-400DRWMLQKGFLKEE 29 1792.8818 1792.881 −0.45 58.2 20614 401-413DLTEKKPWWWHLR 30 1793.9366 1793.9372 0.38 57.6 638701 401-413DLTEKKPWWWHLR 30 1907.9683 1907.9691 0.45 62.1 633624 401-413DLTEKKPWWWHLR 30 2022 2022.001 0.49 67.8 84315

The number PEG molecules per peptide was estimated to be 1.06 for 2-24,0.84 for 9-34, 0.17 for 35-46, 0.26 for 47-78, 0.02 for 79-85 and198-220, 0.54 for 86-106, 0.39 for 245-248, 0.27 for 270-301, 0.21 for321-327, 0.04 for 388-400, and 0.59 for 401-413. Peptide 2-34 wasobserved to have the modification of a 1*linker. Peptide 2-34 wasobserved to have the modification of a 2*linker. Peptide 9-34 wasobserved to have the modification of a 1*linker. Peptide 9-34 wasobserved to have the modification of a 2*linker. Peptide 35-46 wasobserved to have the modification of a 1*linker. Peptide 47-78 wasobserved to have the modification of a 1*linker. Peptide 79-85 wasobserved to have the modification of a 1*linker. Peptide 86-106 wasobserved to have the modification of a 1*linker. Peptide 86-106 wasobserved to have the modification of a 1*linker. Peptide 120-128 wasobserved to have the modification of a 1*oxidation. Peptide 179-197 wasobserved to have the modification of a 1*deamidation. Peptide 198-220was observed to have the modification of a 1*deamidation. Peptide198-220 was observed to have the modification of a 1*linker. Peptide221-233 was observed to have the modification of a 1*deamidation.Peptide 221-233 was observed to have the modification of a1*deamidation. Peptide 245-248 was observed to have the modification ofa 1*linker. Peptide 249-269 was observed to have the modification of a1*oxidation. Peptide 270-301 was observed to have the modification of a1*linker. Peptide 321-327 was observed to have the modification of a1*linker. Peptide 388-400 was observed to have the modification of a1*oxidation. Peptide 388-400 was observed to have the modification of a1*linker. Peptide 401-413 was observed to have the modification of a1*linker. Peptide 401-413 was observed to have the modification of a2*linker.

LC-MS analysis was performed after Asp-N digest of reduced, alkylated,PEGylated 20NHS PEG-htCBS C15S (batch #3). Table 5 shows the relativeabundance of peptides and estimated number of PEGs per peptideidentified by LC/MS/MS for batch #3.

TABLE 5NHS ester PEGylation mapping of Asp-N digest of reduced, alkylated, PEGylated20NHS PEG-htCBS C15S (batch #3) Sequence SEQ ID Theoretical ObservedΔ mass RT Location Sequence NO Mass (Da) Mass (Da) (PPm) (min) Abundance 2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL 2 3294.5865 3294.5829 -1.09 34.3 60072EKGSPE  2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL 2 3408.6182 3408.6154 −0.8237.2 199290 EKGSPE  2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL 2 3522.64993522.6508 0.26 39.7 159538 EKGSPE  9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE 32584.263 2584.2638 0.31 31.3 105797  9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE 32698.2947 2698.294 −0.25 35.2 261221  9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE 32812.3264 2812.3259 −0.18 37.6 96655 35-46 DKEAKEPLWIRP 4 1480.80381480.8039 0.09 51.3 86032 35-46 DKEAKEPLWIRP 4 1594.8355 1594.8362 0.4454 18790 40-46 EPLWIRP 5 909.5072 909.5078 0.62 55 395424 47-78DAPSRCTWQLGRPASESPHHHTAPAK 6 3528.7797 3528.78 0.08 49.6 595498 SPKILP47-78 DAPSRCTWQLGRPASESPHHHTAPAK 6 3642.8114 3642.8084 −0.81 51.7 272886SPKILP 79-85 DILKKIG 7 785.5011 785.5017 0.81 42.5 446682 79-85 DILKKIG7 899.5328 899.5347 2.11 47.7 12609  86-106 DTPMVRINKIGKKFGLKCELL 82459.3756 2459.3745 −0.45 60.7 145186  86-106 DTPMVRINKIGKKFGLKCELL 82573.4073 2573.4088 0.58 64.5 101537  86-106 DTPMVRINKIGKKFGLKCELL 82573.4073 2573.4057 −0.62 67.3 126416 107-119 AKCEFFNAGGSVK 9 1413.67111413.6721 0.71 42.2 506761 120-128 DRISLRMIE 10 1131.607 1131.6075 0.4251.9 725914 120-128 DRISLRMIE 10 1147.6019 1147.602 0.04 44.3 29014133-139 DGTLKPG 11 686.3599 686.3601 0.23 24.5 273520 140-178DTIIEPTSGNTGIGLALAAAVRGYRCII 12 4147.148 4147.1398 -1.97 93.4 188751VMPEKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 13 2098.1647 2098.1663 0.73 65.22212569 179-197 DVLRALGAEIVRTPTNARF 13 2099.1487 2099.1511 1.12 64.7930809 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2597.335 2597.3339 −0.44 58.7645205 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2598.3191 2598.3196 0.2 59.5348068 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2711.3667 2711.3668 0.04 64.431254 221-233 DQYRNASNPLAHY 15 1547.7117 1547.7124 0.48 41.7 119215221-233 DQYRNASNPLAHY 15 1548.6957 1548.6981 1.56 41.1 113439 221-233DQYRNASNPLAHY 15 1548.6957 1548.6959 0.14 42 404271 238-244 DEILQQC 16904.396 904.3962 0.16 33.7 137033 245-248 DGKL 17 431.238 431.2384 0.9320.8 41279 245-248 DGKL 17 545.2697 545.2699 0.44 32.4 38708 249-269DMLVASVGTGGTITGIARKLK 18 2087.1773 2087.1766 −0.32 59.8 378816 249-269DMLVASVGTGGTITGIARKLK 18 2103.1722 2103.1743 1 55.3 81123 270-301EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3632.6975 3632.6934 -1.13 58.1 69301QTTY 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3746.7292 3746.7248 -1.1760 28571 QTTY 302-308 EVEGIGY 20 765.3545 765.3551 0.76 39 267474309-315 DFIPTVL 21 803.4429 803.4434 0.66 66.9 1030604 316-320 DRTW 22588.3231 588.3236 0.72 27.9 474399 321-327 DKWFKSN 23 923.4501 923.4498−0.3 36.3 46084 321-327 DKWFKSN 23 1037.4818 1037.4838 1.93 42.8 12212328-365 DEEAFTFARMLIAQEGLLCGGSAGST 24 3937.9554 3937.9541 −0.33 95.645366 VAVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.875 1735.87 -2.88 4899444 363-375 ELQEGQRCVVILP 26 1539.8079 1539.8083 0.26 56 45283 366-375EGQRCVVILP 27 1169.6227 1169.6233 0.52 54.1 162480 376-387 DSVRNYMTKFLS28 1459.713 1459.7127 −0.16 56.2 715580 388-400 DRWMLQKGFLKEE 291678.8501 1678.8503 0.08 53 430875 388-400 DRWMLQKGFLKEE 29 1694.8451694.8452 0.12 50.2 71467 388-400 DRWMLQKGFLKEE 29 1792.8818 1792.88250.39 58.2 24688 401-413 DLTEKKPWWWHLR 30 1793.9366 1793.9372 0.38 57.7524828 401-413 DLTEKKPWWWHLR 30 1907.9683 1907.969 0.38 62 701248401-413 DLTEKKPWWWHLR 30 2022 2022.0014 0.69 67.6 130215

The number PEG molecules per peptide was estimated to be 1.24 for 2-34,0.98 for 9-34, 0.18 for 35-46, 0.31 for 47-78, 0.03 for 79-85 and198-220, 0.61 for 86-106, 0.48 for 245-248, 0.29 for 270-301, 0.21 for321-327, 0.05 for 388-400, and 0.71 for 401-413.

Peptide 2-34 was observed to have the modification of a 1*Linker.Peptide 2-34 was observed to have the modification of a 2*Linker.Peptide 9-34 was observed to have the modification of a 1*Linker.Peptide 9-34 was observed to have the modification of a 2*Linker.Peptide 35-46 was observed to have the modification of a 1*Linker.Peptide 47-78 was observed to have the modification of a 1*Linker.Peptide 79-85 was observed to have the modification of a 1*Linker.Peptide 86-106 was observed to have the modification of a 1*linker.Peptide 86-106 was observed to have the modification of a 1*linker.Peptide 120-128 was observed to have the modification of a 1*oxidation.Peptide 179-197 was observed to have the modification of a1*deamidation. Peptide 198-220 was observed to have the modification ofa 1*deamidation. Peptide 198-220 was observed to have the modificationof a 1*linker. Peptide 221-233 was observed to have the modification ofa 1*deamidation. Peptide 221-233 was observed to have the modificationof a 1*deamidation. Peptide 245-248 was observed to have themodification of a 1*linker. Peptide 249-269 was observed to have themodification of a 1*oxidation. Peptide 270-301 was observed to have themodification of a 1*linker. Peptide 321-327 was observed to have themodification of a 1*linker. Peptide 388-400 was observed to have themodification of a 1*oxidation. Peptide 388-400 was observed to have themodification of a 1*linker. Peptide 401-413 was observed to have themodification of a 1*linker. Peptide 401-413 was observed to have themodification of a 2*linker.

Seven lysines (K25, K30, K211, K247, K271, K405 and K406) wereunambiguously identified as being PEGylated to a certain extent, out ofwhich K25 and K30 residues seemed the only ones to be always modified tothe full extent among the 3 batches.

Compared to all the previous PEG-htCBS C15S conjugates, the 20NHSPEG-htCBS C15S showed increased viscosity (data not shown) and thusthere was a concern of sufficient free, unreacted PEG removal during thefinal (post PEGylation) buffer exchange. Indeed, processing of theinitial batches (for example LAB and 10L) yielded a large amount ofunreacted PEG carried over to the final product. Corrective measures,which included removal of 20% DMSO from the mixture, higher MWCOmembrane cartridge for TFF unit (100 versus 30 kDa), 3-fold largersurface area and 2-fold dilution of the PEGylation mixture prior bufferexchange, all helped to reduce the viscosity and assisted in betterclearance of free PEG during the final formulation (TR1 and TR2).

Example 5. Attenuation of 200MA PEG-htCBS C15S Efficacy in HO Mice

Repeated administration in HO mice was defined as subcutaneousinjections of 7.5 mg/kg 200MA PEG-htCBS C15S for 5 consecutive daysduring the first dose-week. Dosing was interrupted for a period of 10days and restarted again for an additional 5 consecutive injections inthe second dose-week. Plasma samples were collected before the veryfirst dose for baseline values, 24 hours after the 2nd, 4th, and 5thinjections in both dose-weeks to track the enzyme's efficacy and duringwashout periods to follow recovery of the baseline values (a week afterthe last dose in both dose-weeks, prior to the first dose of thedose-week 2 and two weeks after the very last dose). The study showed inan apparent attenuation of 200MA PEG-htCBS C15S efficacy in thedose-week 2 compared to dose-week 1 (see, Table 7).

A bulkier maleimide PEG molecule did not resolve the attenuation ofPEG-htCBS C15S efficacy in washout experiments. Since the use ofmaleimide PEG, which specifically targets available sulfhydryl residues,was known to yield a well-defined species, the initial approach toreduce the loss of 200MA PEG-htCBS C15S efficacy in washout experimentswas to increase the coverage of the htCBS C15S protein with a bulkiermaleimide PEG. The previously published data showed that PEGylation ofhtCBS with larger maleimide PEG molecule did not affect catalyticactivity in vitro and that 40 kDa linear (ME-400MA) or branched(GL4-400MA) maleimide PEG molecules had similar pharmacokinetics andpharmacodynamics after a single injection in HO mice compared to 200MAPEG-htCBS C15S. See, Bublil et al., J Clin Invest 2016, 126, (6),2372-84 and International application PCT/2016/061050, which are herebyincorporated by reference in their entireties. Table 6 shows that thePEGylation of htCBS C15S does not substantially affect the enzyme'scatalytic activity regardless of the PEG moiety size and the targetgroup.

TABLE 6 CBS specific activities of unmodified htCBS C15S and PEG-htCBSC15S conjugates CBS specific Size & Active moiety activity (U/mg of PEGmoiety structure (target group) protein ± SD) None N/A N/A 1233 ± 201ME-020MA 2 kDa linear maleimide (—SH) 1184 ± 95  ME-200MA0B 20 kDalinear maleimide (—SH) 1339 ± 226 ME-400MA 40 kDa linear maleimide (—SH)1242 ± 158 GL4-400MA 40 kDa 4-arm maleimide (—SH) 1288 ± 221 branchedGL2-800MA 80 kDa 2-arm maleimide (—SH) 1168 ± 134 branched ME-050GS 5kDa linear NHS ester (—NH₂) 1250 ± 52  ME-100GS 10 kDa linear NHS ester(—NH₂) 1233 ± 173 ME-200GS 20 kDa linear NHS ester (—NH₂) 1339 ± 242

The dosing regimen was specifically designed to elicit immune responseto rank conjugates based on their immunogenic properties. The repeateduninterrupted dosing of 200MA PEG-htCBS C15S to homocystinuric HO micehas previously been shown to result in a retained efficacy and asignificant decrease of plasma Hcy levels 24 hours after injection for aperiod of 2 months, although both the baseline level (72 hours postinjection) of plasma Hcy and the peak effect of 200MA PEG-htCBS C15S (24hours post injection) were decreasing over time. The most reasonableexplanation for such attenuation of activity is the host's immuneresponse, which may adversely impact both the efficacy and the safetyand is relatively common among approved therapeutic enzymes. See, Baldoet al., BioDrugs: clinical immunotherapeutics, biopharmaceuticals andgene therapy 2015, 29, (1), 31-55, which is hereby incorporated byreference in its entirety.

The immunogenicity may stem from the trace amounts of unmodified enzymeor insufficiently masked htCBS C15S with site selective maleimide PEGmolecule conjugation. Therefore, a complete absence of unmodified enzymeand more generous masking of potential immunogenic epitopes of PEG-htCBSC15S conjugate from the immune system by increased PEGylation would beexpected to prevent the decrease in efficacy.

Example 6. Retaining Efficacy of 20NHS PEG-htCBS in HO Mice

NHS ester PEG-htCBS C15S conjugates modified with ME-050GS (5NHSPEG-htCBS C15S) and ME-200GS (20NHS PEG-htCBS C15S) were analyzed inwashout experiments, and results were compared to results from similaranalysis of maleimide PEGylated conjugates. Table 7 provides the percentchanges in metabolites resulting from administration of differentPEG-htCBS C15S conjugates over 3 weeks. (W1=1 week, W2=2 weeks, and W3=3weeks)

TABLE 7 Maximal changes in the plasma sulfur amino acid levels aftertreatment in HO mice with PEGylated htCBS C15S conjugates Hcy decreaseat trough (%) Cth increase at peak (%) Cys increase at peak (%) PEGmolecule W1 W2 W3 W1 W2 W3 W1 W2 W3 PBS (control) −8.0 −8.1 n/a −1 −19n/a −18.1 4.7 n/a ME-400MA 73.2 64.9 58.6 383 329 365 17.2 6.3 8.0GL4-400MA 74.6 41.1 34.0 715 723 449 97.8 47.5 51.8 ME-400MA 74.9 52.041.5 696 649 443 71.9 44.3 43.6 ME-050GS 74.8 58.5 42.1 978 795 833 70.775.0 58.3 ME-200GS 76.9 70.2 65.8 676 492 432 58.7 60.6 50.8 ME-200MA68.0 24.0 n/a 796 508 n/a 35.7 16.1 n/a ME-200MA 82.0 56.6 50.4 700 866335 71.5 37.9 31.5 ME-200MA 80.4 60.6 50.1 1023 764 1100 76.6 77.3 58.3

The 5NHS PEG-htCBS C15S showed an excellent pharmacodynamics responseafter the first set of injections but a significant loss of efficacy wasobserved in the subsequent dose-weeks for Hcy. Hcy levels dropped to48/80/111 μM (baseline levels: 192/175/201 μM) with a simultaneousincrease in Cth levels to 43/35/37 μM (baseline levels: 4/4/4 μM) andconcurrent increase of Cys levels to 219/225/174 μM (baseline levels:129/137/146 μM) in dose-weeks 1/2/3 (FIG. 6 and Table 7).

20NHS PEG-CBS showed a balanced response in all three dose-weeks: theplasma Hcy concentration dropped to 38/49/56 μM from baseline levels164/165/143 μM with a subsequent increase in Cth levels to 40/30/27 μM(baseline levels: 5/4/4 μM) and concurrent increase of Cys levels to234/237/222 μM (baseline levels: 147/149/148 μM) in dose-weeks 1/2/3. Asa control experiment, two additional batches of 200MA PEG-htCBS C15Swere analyzed in a washout experiment and attenuated efficacy insubsequent dose-weeks was again observed: plasma Hcy dropped to44/88/112 μM (baseline levels: 224/215/224 μM) with consequent increasein Cth levels to 40/31/43 μM (baseline levels: 4/3/4 μM) and concurrentincrease of Cys levels to 219/219/196 μM (baseline levels: 124/118/144μM) in dose-weeks 1/2/3. The 20NHS PEG-CBS showed the least attenuationof the pharmacodynamic effect on plasma metabolites after repeatedadministration of a PEGylated enzyme to HO mice (Table 7).

Example 7. Rescuing the Phenotype of KO Mice by Treatment with 20NHSCBS-PEG

To assess the potentially beneficial effects of 20NHS PEG-htCBS C15Streatment on overall health and thriving of KO mice.

The body weights and weight gains of 20NHS PEG-htCBS C15S-treated mice(n-16M+11F) were determined every other day from weaning (day 21) up to35 days of age and compared to sex- and age-matched PBS-treatedheterozygous+/−mice (n-14M+13F). Mice received subcutaneous injections3× a week of 7.5 mg/kg from day 2 of age. Body weight (FIG. 7A) andweight gain (FIG. 7B) of 20NHS PEG-htCBS C15S-treated CBS KO male (graysolid line & circles) and female (gray dashed line & circles) mice arecompared to PBS-injected+/−males (black solid line & squares) andfemales (black dashed line & squares).

At the time of weaning (day 21), no significant differences were foundin body weights between both heterozygous or KO mice and males orfemales receiving either PBS or 20NHS PEG-htCBS C15S except for malesversus females on 20NHS PEG-htCBS C15S injections (p<0.01). Two dayslater (day 23) no significant difference in body weights among studymice was found. From day 25 on, the +/−males gained weight significantly(p<0.001) more and thus increase their body weight more than the+/−females as well as the 20NHS PEG-htCBS C15S-treated KO males orfemales. In KO mice, weaning led to a brief period of slower growth(days 23 and 25) characterized by a smaller weight gain and practicallymaintained body weight followed by steady gains in weight. Weight gainsof KO males and females treated with 20NHS PEG-htCBS C15S wereessentially identical and at days 33 and 35 their weight gains and bodyweights of treated KO females caught up with those of PBS-injectedheterozygous females, while KO males fell behind a bit theirheterozygous counterparts. Taken together, 20NHS PEG-htCBS C15S-treatedKO mice gained weight steadily with females more than males catching upwith the PBS-treated heterozygous mice in terms of body weight at theend of the 5-week treatment period.

Example 8. Sustaining Improved Plasma Levels by Treatment with 20NHSPEG-htCBS C15S

Clinical symptoms of the I278T mice, such as facial alopecia orosteoporosis, take time to develop as well as get corrected withtreatment. In this Example, treatment of I278T mice with 20NHS PEG-htCBSC15S was shown to significantly improve or normalize the underlyingmetabolic imbalance and sustain it over a longer period. FIG. 8A andFIG. 8B show sustained efficacy of 20NHS PEG-htCBS C15S on plasma sulfuramino acids in the I278T mouse model. FIG. 8A showed a sustainedimprovement in plasma Hcy, Cth, and Cys levels in asymptomatic weanedI278T mice of 3 weeks of age (n=3) treated since then with three weeklySC injections (7.5 mg/kg) for a period of about 9 months. The initiallevels recorded prior the first dose at day 21 showed highly elevatedHcy (288 μM), roughly half of the normal Cys levels (129 μM) and lowplasma Cth (1.4 μM). Plasma metabolites determined 72 hours after the3rd (day 28) and the 18th dose (day 63), respectively, illustrated thatthe lack of dosing during weekends resulted in somewhat a similarprofile compared to the initial levels except for Cth, a marker of 20NHSPEG-htCBS C15S activity, for which elevated plasma concentrationpersisted most likely due to its slow renal clearance.

On the contrary, plasma levels determined 24 hours after 20NHS PEG-htCBSC15S administration (e.g. at day 64 and applies for all the subsequenttime points) demonstrate substantially and significantly decreased Hcylevels (105 μM, p<0.001), normalized plasma Cys (209 μM, p<0.001) andelevated Cth (43 μM, p<0.001) compared to the initial values. Similarly,FIG. 8B exemplifies a persistent correction of plasma sulfur amino acidsin 26 weeks old I278T mice (n=10) showing multiple clinical signs of thediseases and treated since then (3× a week, SC, 7.5 mg/kg) for a periodof about 6 months. The initial levels recorded prior the first dose atday 182 showed highly elevated Hcy (321 μM), roughly half of the normalCys levels (127 μM) and low plasma Cth (0.3 μM). The 24 hours afterinitial administration of 20NHS PEG-htCBS C15S (day 184) resulted in amarked and significant decrease of plasma Hcy (143 μM, p<0.001),normalization of plasma Cys (246 μM, p<0.001) and elevation of Cth (54μM, p<0.001) compared to the pre-treatment values. The improved plasmametabolite profile was maintained with regular ERT administration for aperiod of about 6 months. Taken together, regular SC injections of 20NHSPEG-htCBS C15S yielded a sustained improved plasma metabolite profile inthe I278T mouse, thus permitting efficacy studies requiring long-termadministration.

Example 9. Normalizing Tissue Metabolite Levels and Improving theBalance of Metabolites

Administration of 20NHS PEG-htCBS C15S was observed to result incorrection of tissue metabolites, which correlated to improved plasmametabolites balance in adult I278T mice. Concentrations ofsulfur-containing metabolites in liver, kidney and brain homogenates andcorresponding plasma levels of about 2-month-old I278T mice (n=3)treated with 20NHS PEG-htCBS C15S (SC, 7.5 mg/kg) for a period of 3weeks compared to age-matched healthy heterozygous mice (n=3) as well asuntreated I278T mice (n=3) were measured. The sulfur-containingmetabolites included homocysteine (Hcy), cysteine (Cys), cystathionine(Cth), homolanthionine (Hlth), and lanthionine (Lth). Total homocysteineand cysteine was determined in plasma, while nonprotein-bound fractionsof these thiols were measured in tissue homogenates. The I278Tadolescent mice (n=3) were treated for a period of 3 weeks with 20NHSPEG-htCBS C15S (3× a week, SC, 7.5 mg/kg) and compared to age-matcheduntreated I278T mice (n=3) and untreated healthy heterozygous controls(n=3). Data were compared using ANOVA followed by Tukey's post hoc testto determine significance: * p<0.05, ** p<0.01, *** p<0.001,ns—non-significant. Results are described by μM in nmol/g of tissue ornM in pmol/g of tissue.

Total Hcy and non-protein bound Hcy was greatly elevated in plasma andliver/kidney/brain homogenates, respectively, in the untreated I278Tmice (310 μM in plasma and 29/21/12 μM in nmols/g of liver/kidney/brain)compared to both heterozygous controls (12 μM in plasma and 8/6/4 μM innmols/g of liver/kidney/brain) and 20NHS PEG-htCBS C15S-treated I278Tmice (54 μM in plasma and 8/4/1 μM in nmols/g of liver/kidney/brain,p<0.001 for plasma and <0.01 for tissues). The 4.5-fold elevated totalHcy in plasma, non-protein bound Hcy in tissues of the treated I278Tmice was not significantly different from the levels in healthyheterozygous mice.

Total Cys was substantially decreased in plasma (91 μM), but thenon-protein bound Cys in liver/kidney/brain homogenates of the untreatedI278T mice (21/432/44 μM in nmols/g of liver/kidney/brain) was notobserved to be different compared to the 20NHS PEG-htCBS C15S-treatedI278T mice (224 μM in plasma and 36/265/51 μM in nmols/g ofliver/kidney/brain, p<0.01 for plasma and non-significant for tissues).The Cys levels in the treated I278T mice were similar to those ofhealthy heterozygous controls (255 μM in plasma and 81/322/44 μM innmols/g of liver/kidney/brain, p=non-significant).

As anticipated, Cth levels in both plasma and tissues of untreated I278Tmice were diminished: 0.3 μM in plasma and 0.4/1.2/0.8 μM in nmols/g ofliver/kidney/brain. Treatment with 20NHS PEG-htCBS C15S resulted insignificant elevation of Cth in plasma (39 μM) and kidney homogenate (36μM in nmols/g of tissue) and lesser increase in liver and brainhomogenates (5 and 3 μM in nmols/g of tissue) compared to the untreatedI278T mice (p<0.05 for both plasma and tissue homogenates). Althoughplasma and kidney levels of Cth were substantially higher in the treatedI278T mice in comparison to healthy heterozygous controls (1 μM inplasma, p<0.01 and 1.2 μM in nmols/g in kidney, p<0.01) likely due toactivity of 20NHS PEG-htCBS C15S in circulation, they were not quitenormalized to healthy levels in liver (20 μM in nmols/g of tissue,non-significant) and brain homogenates (8 μM in nmols/g of tissue,p<0.05).

No significant differences were observed in plasma and tissue levels ofMet or GSH among the untreated I278T mice, the 20NHS PEG-htCBSC15S-treated I278T mice and healthy heterozygous controls.

Levels of thioethers homolathionine (Hlth) and lanthionine (Lth) inplasma and tissue homogenates have also been identified as emergingsurrogate markers of H₂S biogenesis. The levels of Hlth were markedlyelevated in plasma (143 nM) and liver/kidney/brain homogenates of theuntreated I278T mice (6351/3324/1065 nM in pmol/g of tissue) and the20NHS PEG-htCBS C15S treatment resulted in substantial correction ofthese levels to 45 nM in plasma (non-significant) and 170/1317/520 nM inpmols/g of liver/kidney/brain tissue (p<0.05/0.05/0.01). Interestingly,while liver Hlth in healthy heterozygous mice was significantly higher(996 nM in pmol/g of tissue, p<0.01) compared to the treated I278T mice,the treatment resulted just in partial reduction of Hlth levels in theremaining compartments compared to those found in heterozygous mice: 8nM in plasma (p<0.05) and 247/22 nM in pmol/g of kidney/brain tissues(p<0.01/0.001).

Regarding the second thioether Lth, only the plasma levels weresignificantly elevated in treated I278T compared to the untreated ones(75 versus 17 nM, p<0.01), while levels of Lth in tissue homogenatesremained similar (21/113/110 versus 14/92/104 nM in pmols/g ofliver/kidney/brain tissue) due to low tissue permeability or a lack of aspecific transporter for Lth. Plasma Lth in healthy heterozygous micewas significantly lower (32 nM, p<0.01), while its tissue levels wereeither elevated in liver (122 nM in pmols/g of tissue, p<0.05) and brain(304 nM in pmols/g of tissue, p<0.001) or remained similar in kidney(145 nM in pmols/g of tissue) when compared to the levels observed inthe treated I278T mice. Therefore, administration of 20NHS PEG-htCBSC15S improved or restored metabolic balance in plasma and tissues of thetreated I278T mice.

Example 10. Comparing Methylation Capacity Among KO, HO, and I278T MiceTreated with 20NHS PEG-htCBS CBS

Plasma and tissue levels of the SAM/SAH ratio in I278T and KO mice werecompared to the ratio in healthy heterozygous control mice and untreatedmice. The I278T adolescent mice (n=3) were treated for a period of 3weeks with PEG-htCBS C15S (3× a week, SC, 7.5 mg/kg) and compared toage-matched untreated I278T mice (n=3) and untreated healthyheterozygous controls (n=3). Treated KO mice were dosed 3× a week 7.5mg/kg from day 2 of age. Each group consisted of at least three 18 daysold mice. All data were compared using multivariate ANOVA followed byTukey's post hoc test to determine significance: * p<0.05, ** p<0.01,*** p<0.001, ns—non-significant.

Methylation capacity of the untreated I278T mice was substantiallydecreased based on SAM/SAH ratio in plasma (0.4) and liver/kidney/brainhomogenates (0.3/0.4/0.2) and was substantially ameliorated to a ratioof 3 in plasma (p<0.001) and 0.7/2.1/1.5 in liver/kidney/brainhomogenates (non-significant for liver; but p<0.01 for both kidney andbrain tissue) in the treated group. The improved methylation capacity ofthe 20NHS PEG-htCBS C15S-treated I278T mice approached or evennormalized the one determined in healthy heterozygous mouse plasmas (5,p<0.01) and liver/kidney/brain homogenates (1.3/3.9/2.0, non-significantfor liver and brain, but p<0.05 for kidney).

Administration of 20NHS PEG-htCBS C15S was also observed tosignificantly improve the SAM/SAH ratio in plasma and tissues of thetreated KO mice compared to the untreated ones but did not normalize itto the levels seen in healthy heterozygous controls. The SAM/SAH ratiowas quite small in the livers of WT controls compared to plasma andother tissue levels and, more importantly, comparable to the levelsfound in KO mice (0.78 and 0.94 in control healthy heterozygous and KO,p=ns). In addition, the 20NHS PEG-htCBS C15S treatment further decreasedthe SAM/SAH ratio in the treated KO mice compared to untreated onessignificantly (0.28, p<0.01). Therefore, administration of 20NHSPEG-htCBS C15S improved or restored the metabolic balance in plasma andtissues of the treated KO mice.

Example 11. Pharmacokinetics of 20NHS PEG-htCBS C15S in Rats

Pharmacokinetic properties of 20NHS PEG-htCBS C15S were also determinedin wild-type Sprague Dawley rats. CBS specific activity in plasma ofmale (black, n=11 each group) and female rats (gray, n=8 each group)after a single SC administration of 8 mg/kg (solid lines) or 24 mg/kg20NHS PEG-htCBS C15S (dashed lines) in log scale is shown in FIG. 9A.

The bioavailability of 20NHS PEG-htCBS C15S was observed to be nearlyidentical at the different doses (8 and 24 mg/kg) suggesting that theobserved differences in absorption in rats were likely due to sexualdimorphism. The elimination phase of 20NHS PEG-htCBS C15S was log-linearin all cohorts and conformed to first order kinetics meaning that thetime required for half the concentration of 20NHS PEG-htCBS C15Sactivity to clear from the plasma was constant. The mean value ofelimination half-life across groups was 42±2 hours. Despite differencesbetween male and female rats, the PK parameters estimated from plasmalevels of 20NHS PEG-htCBS C15S activity after SC dosing were doseproportional to 24 mg/kg.

Differences due to sexual dimorphism in rats were also observed in arepeated dosing study, where the rats received a total of 9 injections48 hours apart of 4, 8, and 24 mg/kg 20NHS PEG-htCBS C15S (FIG. 9B andFIG. 9C). In general, initial as well as steady state levels of 20NHSPEG-htCBS C15S activity in plasma were higher in female than male ratsat the same dose level correlating with the observation for the singledose study. Plasma levels of 20 NHS PEG-htCBS C15S after repeated dosingdemonstrated little or no accumulation at any dose level, even thoughthe doses were administered when only slightly more than half of the20NHS PEG-htCBS C15S have cleared from the plasma (48 hours dosinginterval versus average t_(1/2-E)=42 hours). To aid in the understandingof these unexpected observations, the approach to steady state and thepredicted steady state plasma levels were calculated after hypotheticalrepeated dosing using the simulation function in the PK software. Aftermultiple doses, the observed plasma levels of 20NHS PEG-htCBS C15Sactivity were 15-52% and 0-35% of the predicted values for the peaks andtroughs at steady state, as shown in Table 8. (N/A=not applicable)

TABLE 8 Predicted and observed c_(max-SS) and c_(min-sS) Male (8 mg/kg)Female (8 mg/kg) Male (24 mg/kg) Female (24 mg/kg) PK parameter ObservedPredicted Observed Predicted Observed Predicted Observed Predictedc_(max) (mU/μl) 26 N/A 43 N/A 110 N/A 167 N/A c_(max-SS) (mU/μl) 10 6642 127 99 227 196 378 c_(min-SS) (mU/μl) 0 41 3 61 60 171 78 249Observed versus predicted 20NHS PEG−htCBS C15S plasma activity at steadystate (% or predicted value) c_(max-SS) 15 33 44 52 c_(min-SS) 0 5 35 31

The observed c_(max) and c_(min) correspond to the highest and lowestconcentration of 20NHS PEG-htCBS C15S observed in plasma after the 2nddose. The predicted values are from simulations using thetime-concentration curves from the single dose PK study. The plasmalevels after repeated dosing were observed to be closer to theirpredicted values at 24 mg/kg dose than at 8 mg/kg dose in both sexes.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a”, “an”, and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or the entiregroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyantibiotic, therapeutic or active ingredient; any method of production;any method of use; etc.) can be excluded from any one or more claims,for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

1. (canceled)
 2. A method of PEGylating a human truncated cystathionineβ-synthase (CBS) protein containing a mutation of a cysteine to a serineat amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBSC15S), the method comprising: (a) conjugating the htCBS C15S with one ora plurality of NHS ester PEG molecules in solution at a molar excess ofthe NHS ester PEG molecules less than about 40-fold to create a batch,wherein the plurality of NHS ester PEG molecules are 5 kDa, 10 kDa, or20 kDa NHS ester PEG molecules; (b) comparing a retention time fromnon-reduced capillary electrophoresis (NR-CE) analysis of the batch to aretention time from NR-CE analysis of htCBS C15S with acceptablePEGylation to identify insufficient PEGylation, wherein the batch withinsufficient PEGylation has a retention time less than the retentiontime of the htCBS C15S with acceptable PEGylation; and (c) addingadditional NHS ester PEG molecules to the batch to reduce the amount ofinsufficient PEGylation in the batch, thereby PEGylating the htCBS C15S.3. The method of claim 2, wherein the NHS ester PEG molecules arepresent in a molar excess of about 10-fold.
 4. The method of claim 2,wherein the NHS ester PEG molecules are present in a molar excess ofabout 20-fold.
 5. The method of claim 2, wherein the NHS ester PEGmolecules are present in a molar excess less than 20-fold, less than10-fold, less than 5-fold, or less than 2-fold.
 6. The method of claim2, wherein the one or the plurality of NHS ester PEG molecules consistsof 5 kDa NHS ester (5NHS) PEG molecules.
 7. The method of claim 2,wherein the one or the plurality of NHS ester PEG molecules consists of10 kDa NHS ester (10NHS) PEG molecules.
 8. The method of claim 2,wherein the one or the plurality of NHS ester PEG molecules consists of20 kDa NHS ester (20NHS) PEG molecules.
 9. The method of claim 2,wherein each of the plurality of NHS ester PEG molecules is less thanabout 20 kDa.
 10. The method of claim 2, wherein htCBS C15S withinsufficient PEGylation comprises fewer than 15, fewer than 10, fewerthan 5, or fewer than 1 PEGylated amino acid.
 11. The method of claim 2,wherein htCBS C15S with acceptable PEGylation comprises at least 1PEGylated amino acid. 12.-25. (canceled)
 26. The method of claim 2,wherein htCBS C15S with acceptable PEGylation comprises at least 5PEGylated sites.